Recombinant adenoviral vector and methods of use

ABSTRACT

This invention provides a recombinant adenovirus expression vector characterized by the partial or total deletion of the adenoviral protein IX DNA and having a gene encoding a foreign protein or a functional fragment or mutant thereof. Transformed host cells and a method of producing recombinant proteins and gene therapy also are included within the scope of this invention.  
     Thus, for example, the adenoviral vector of this invention can contain a foreign gene for the expression of a protein effective in regulating the cell cycle, such as p53, Rb, or mitosin, or in inducing cell death, such as the conditional suicide gene thymidine kinase. (The latter must be used in conjunction with a thymidine kinase metabolite in order to be effective).

[0001] This application is a continuation-in-part of U.S. Ser. No.08/233,777, filed May 19, 1994, which is a continuation-in-part of U.S.Ser. No. 08/142,669 filed Oct. 25, 1993, the contents of which arehereby incorporated by reference into the present disclosure.

BACKGROUND OF THE INVENTION

[0002] Throughout this application, various publications are referred toby citations within parentheses and in the bibliographic description,immediately preceding the claims. The disclosures of these publicationsare hereby incorporated by reference into the present disclosure to morefully describe the state of the art to which this invention pertains.

[0003] Production of recombinant adenoviruses useful for gene therapyrequires the use of a cell line capable of supplying in trans the geneproducts of the viral E1 region which are deleted in these recombinantviruses. At present the only useful cell line available is the 293 cellline originally described by Graham et al. in 1977. 293 cells containapproximately the left hand 12% (4.3 kb) of the adenovirus type 5 genome(Aiello (1979) and Spector (1983)).

[0004] Adenoviral vectors currently being tested for gene therapyapplications typically are deleted for Ad2 or Ad5 DNA extending fromapproximately 400 base pairs from the 5′ end of the viral genome toapproximately 3.3 kb from the 5′ end, for a total E1 deletion of 2.9 kb.Therefore, there exists a limited region of homology of approximately 1kb between the DNA sequence of the recombinant virus and the Ad5 DNAwithin the cell line. This homology defines a region of potentialrecombination between the viral and cellular adenovirus sequences. Sucha recombination results in a phenotypically wild-type virus bearing theAd5 E1 region from the 293 cells. This recombination event presumablyaccounts for the frequent detection of wild-type adenovirus inpreparations of recombinant virus and has been directly demonstrated tobe the cause of wild-type contamination of the Ad2 based recombinantvirus Ad2/CFTR-1 (Rich et al. (1993)).

[0005] Due to the high degree of sequence homology within the type Cadenovirus subgroup such recombination is likely to occur if the vectoris based on any group C adenovirus (types 1, 2, 5, 6).

[0006] In small scale production of recombinant adenoviruses, generationof contaminating wild-type virus can be managed by a screening processwhich discards those preparations of virus found to be contaminated. Asthe scale of virus production grows to meet expected demand for genetictherapeutics, the likelihood of any single lot being contaminated with awild-type virus also will rise as well as the difficulty in providingnon-contaminated recombinant preparations.

[0007] There will be over one million new cases of cancer diagnosed thisyear, and half that number of cancer-related deaths (American CancerSociety, 1993). p53 mutations are the most common genetic alterationassociated with human cancers, occurring in 50-60% of human cancers(Hollstein et al. (1991); Bartek et al. (1991); Levine (1993)). The goalof gene therapy in treating p53 deficient tumors, for example, is toreinstate a normal, functional copy of the wild-type p53 gene so thatcontrol of cellular proliferation is restored. p53 plays a central rolein cell cycle progression, arresting growth so that repair or apoptisiscan occur in response to DNA damage. Wild-type p53 has recently beenidentified as a necessary component for apoptosis induced by irradiationor treatment with some chemotherapeutic agents (Lowe et al. (1993) A andB). Due to the high prevalence of p53 mutations in human tumors, it ispossible that tumors which have become refractory to chemotherapy andirradiation treatments may have become so due in part to the lack ofwild-type p53. By resupplying functional p53 to these tumors, it isreasonable that they now are susceptible to apoptisis normallyassociated with the DNA damage induced by radiation and chemotherapy.

[0008] One of the critical points in successful human tumor suppressorgene therapy is the ability to affect a significant fraction of thecancer cells. The use of retroviral vectors has been largely exploredfor this purpose in a variety of tumor models. For example, for thetreatment of hepatic malignancies, retroviral vectors have been employedwith little success because these vectors are not able to achieve thehigh level of gene transfer required for in vivo gene therapy (Huber, B.E. et al., 1991; Caruso M. et al., 1993).

[0009] To achieve a more sustained source of virus production,researchers have attempted to overcome the problem associated with lowlevel of gene transfer by direct injection of retroviral packaging celllines into solid tumors (Caruso, M. et al., 1993; Ezzidine, Z. D. etal., 1991; Culver, K. W. et al., 1992). However, these methods areunsatisfactory for use in human patients because the method istroublesome and induces an inflammatory response against the packagingcell line in the patient. Another disadvantage of retroviral vectors isthat they require dividing cells to efficiently integrate and expressthe recombinant gene of interest (Huber, B. E. 1991). Stable integrationinto an essential host gene can lead to the development or inheritanceof pathogenic diseased states.

[0010] Recombinant adenoviruses have distinct advantages over retroviraland other gene delivery methods (for review, see Siegfried (1993)).Adenoviruses have never been shown to induce tumors in humans and havebeen safely used as live vaccines (Straus (1984)). Replication deficientrecombinant adenoviruses can be produced by replacing the E1 regionnecessary for replication with the target gene. Adenovirus does notintegrate into the human genome as a normal consequence of infection,thereby greatly reducing the risk of insertional mutagenesis possiblewith retrovirus or adeno-associated viral (AAV) vectors. This lack ofstable integration also leads to an additional safety feature in thatthe transferred gene effect will be transient, as the extrachromosomalDNA will be gradually lost with continued division of normal cells.Stable, high titer recombinant adenovirus can be produced at levels notachievable with retrovirus or AAV, allowing enough material to beproduced to treat a large patient population. Moreover, adenovirusvectors are capable of highly efficient in vivo gene transfer into abroad range of tissue and tumor cell types. For example, others haveshown that adenovirus mediated gene delivery has a strong potential forgene therapy for diseases such as cystic fibrosis (Rosenfeld et al.(1992); Rich et al. (1993)) and α₁-antitrypsin deficiency (Lemarchand etal. (1992)). Although other alternatives for gene delivery, such ascationic liposome/DNA complexes, are also currently being explored, noneas yet appear as effective as adenovirus mediated gene delivery.

[0011] As with treating p53 deficient tumors, the goal of gene therapyfor other tumors is to reinstate control of cellular proliferation. Inthe case of p53, introduction of a functional gene reinstates cell cyclecontrol allowing for apoptotic cell death induced by therapeutic agents.Similarly, gene therapy is equally applicable to other tumor suppressorgenes which can be used either alone or in combination with therapeuticagents to control cell cycle progression of tumor cells and/or inducecell death. Moreover, genes which do not encode cell cycle regulatoryproteins, but directly induce cell death such as suicide genes or, geneswhich are directly toxic to the cell can be used in gene therapyprotocols to directly eliminate the cell cycle progression of tumorcells.

[0012] Regardless of which gene is used to reinstate the control of cellcycle progression, the rationale and practical applicability of thisapproach is identical. Namely, to achieve high efficiencies of genetransfer to express therapeutic quantities. of the recombinant product.The choice of which vector to use to enable high efficiency genetransfer with minimal risk to the patient is therefore important to thelevel of success of the gene therapy treatment.

[0013] Thus, there exists a need for vectors and methods which providehigh level gene transfer efficiencies and protein expression whichprovide safe and effective gene therapy treatments. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0014] This invention provides a recombinant adenovirus expressionvector characterized by the partial or total deletion of the adenoviralprotein IX DNA and having a gene encoding a foreign protein or afunctional fragment or mutant thereof. Transformed host cells and amethod of producing recombinant proteins and gene therapy also areincluded within the scope of this invention.

[0015] Thus, for example, the adenoviral vector of this invention cancontain a foreign gene for the expression of a protein effective inregulating the cell cycle, such as p53, Rb, or mitosin, or in inducingcell death, such as the conditional suicide gene thymidine kinase. (Thelatter must be used in conjunction with a thymidine kinase metabolite inorder to be effective).

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 shows a recombinant adenoviral vector of this invention.This construct was assembled as shown in FIG. 1. The resultant virusbears a 5′ deletion of adenoviral sequences extending from nucleotide356 to 4020 and eliminates the E1a and E1b genes as well as the entireprotein IX coding sequence, leaving the polyadenylation site shared bythe E1b and pIX genes intact for use in terminating transcription of anydesired gene.

[0017]FIG. 2 shows the amino acid sequence of p110^(RB).

[0018]FIG. 3 shows a DNA sequence encoding a retinoblastoma tumorsuppressor protein.

[0019]FIG. 4 shows schematic of recombinant p53/adenovirus constructswithin the scope of this invention. The p53 recombinants are based on Ad5 and have had the E1 region of nucleotides 360-3325 replaced with a 1.4kb full length p53 cDNA driven by the Ad 2 MLP (A/M/53) or human CMV(A/C/53) promoters followed by the Ad 2 tripartite leader cDNA. Thecontrol virus A/M has the same Ad 5 deletions as the A/M/53 virus butlacks the 1.4 kb p53 cDNA insert. The remaining E1b sequence (705nucleotides) have been deleted to create the protein IX deletedconstructs A/M/N/53 and A/C/N/53. These constructs also have a 1.9 kbXba I deletion within adenovirus type 5 region E3.

[0020]FIGS. 5A and 5B show p53 protein expression in tumor cellsinfected with A/M/53 and A/C/53. FIG. 5A) Saos-2 (osteosarcoma) cellswere infected at the indicated multiplicities of infection (MOI) witheither the A/M/53 or A/C/53 purified virus and harvested 24 hours later.The p53 antibody pAb 1801 was used to stain immunoblots of samplesloaded at equal total protein concentrations. Equal proteinconcentration of SW480 cell extracts, which overexpress mutant p53protein, were used as a marker for p53 size. “O” under the A/C/53heading indicates a mock infection, containing untreated Saos-2 lysate.FIG. 5B) Hep 3B (hepatocellular carcinoma) cells were infected with theA/M/53 or A/C/53 virus at the indicated MOI and analyzed as in part A.)The arrow indicates the position of the p53 protein.

[0021]FIGS. 6A through 6C show p53 dependent Saos-2 morphology change.Subconfluent (1×10⁵ cells/10 cm plate) Saos-2 cells were eitheruninfected (A), infected at an MOI=50 with (B) the control A/M virus or(C) the A/C/53 virus. The cells were photographed 72 hourspost-infection.

[0022]FIG. 7 shows p53 dependent inhibition of DNA synthesis in humantumor cell lines by A/M/N/53 and A/C/N/53. Nine different tumor celllines were infected with either control adenovirus A/M (-x-x-), or thep53 expressing A/M/N/53 (-Δ-Δ-), or A/C/N/53 (-∘-∘-) virus at increasingMOI as indicated. The tumor type and p53 status is noted for each cellline (wt=wild type, null=no protein expressed, mut=mutant proteinexpressed). DNA synthesis was measured 72 hours post-infection asdescribed below in Experiment No. II. Results are from triplicatemeasurements at each dose (mean+/−SD), and are plotted as % of mediacontrol versus MOI. * H69 cells were only tested with A/M and A/M/N/53virus.

[0023]FIG. 8 shows tumorigenicity of p53 infected Saos-2 cells in nudemice. Saos-2 cells were infected with either the control A/M virus orthe p53 recombinant A/M/N/53 at MOI=30. Treated cells were injectedsubcutaneously into the flanks of nude mice, and tumor dimensions weremeasured (as described in Experiment No. II) twice per week for 8 weeks.Results are plotted as tumor size versus days post tumor cellimplantation for both control A/M (-x-x-) and A/M/N/53 (-Δ-Δ-) treatedcells. Error bars represent the mean tumor size=/−SEM for each group of4 animals at each time point.

[0024]FIG. 9 is expression of rAd/p53 RNA in established tumors. H69(SCLC) cells were injected subcutaneously into nude mice and allowed todevelop tumors for 32 days until reaching a size of approximately 25-50mm³. Mice were randomized and injected peritumorally with 2×10⁹ pfu ofeither control A/C/β-gal or A/C/53 virus. Tumors were excised 2 and 7days post injection, and polyA RNA was prepared from each tumor sample.RT-PCR was carried out using equal RNA concentrations and primersspecific for recombinant p53 message. PCR amplification was for 30cycles at 94° C. 1 min., 55° C. 1.5 min., 72° C. 2 min., and a 10 min.,72° C. final extension period in an Omnigen thermalcycler (Hybaid). ThePCR primers used were a 5′ Tripartite Leader cDNA(5′-CGCCACCGAGGGACCTGAGCGAGTC-3′) and a 3′ p53 primer(5′-TTCTGGGAAGGGACAGAAGA-3′). Lanes 1, 2, 4, and 5 are p53 treatedsamples excised at day 2 or 7 as indicated. Lanes 3 and 6 are from β-galtreated tumors. Lanes 7,8, and 9 are replicates of lanes 4,5, and 6respectively, amplified with actin primers to verify equal loading. Lane10 is a positive control using a tripartite/p53 containing plasmid.

[0025]FIGS. 10A and 10B show in vivo tumor suppression and increasedsurvival time with A/M/N/53. H69 (SCLC) tumor cells were injectedsubcutaneously into nude mice and allowed to develop for 2 weeks.Peritumoral injections of either buffer alone ( - - - ), control A/Madenovirus (-x-x-), or A/M/N/53 (-Δ-Δ), both viruses (2×10⁹pfu/injection) were administered twice per week for a total of 8 doses.Tumor dimensions were measured twice per week and tumor volume wasestimated as described in Experiment No. II. A) Tumor size is plottedfor each virus versus time (days) post inoculation of H69 cells. Errorbars indicate the mean tumor size +/−SEM for each group of 5 animals.Arrows indicate days virus injections. B). Mice were monitored forsurvival and the fraction of mice surviving per group versus time postinoculation of buffer alone ( - - - - ), control A/M ( . . . . . . . . .) or A/M/N/53 (-) virus treated H69 cells is plotted.

[0026]FIGS. 11A through 11C show maps of recombinant plasmidconstructions. Plasmids were constructed as detailed in below. Boldlines in the constructs indicate genes of interest while boldface typeindicates the restriction sites used to generate the fragments to beligated together to form the subsequent plasmid as indicated by thearrows. In FIG. 11A, the plasmid pACNTK was constructed by subcloningthe HSV-TK gene from pMLBKTK (ATCC No. 39369) into the polylinker of acloning vector, followed by isolation of the TK gene with the desiredends for cloning into the pACN vector. The pACN vector containsadenoviral sequences necessary for in vivo recombination to occur toform recombinant adenovirus (see FIG. 12). In FIG. 11B, the constructionof the plasmid pAANTK is shown beginning with PCR amplified fragmentsencoding the α-fetoprotein enhancer (AFP-E) and promoter (AFP-P) regionssubcloned through several steps into a final plasmid where the AFPenhancer and promoter are upstream of the HSV-TK gene followed byadenovirus Type 2 sequences necessary for in vivo recombination to occurto form recombinant adenovirus. In FIG. 11C, the construction of theplasmid pAANCAT is shown beginning with the isolation of thechloramphenicol acetyltransferase (CAT) gene from a commerciallyavailable plasmid and subcloning it into the pAAN plasmid (see above),generating the final plasmid pAANCAT where the AFP enhancer/promoterdirect transcription of the CAT gene in an adenovirus sequencebackground.

[0027]FIG. 12 is a schematic map of recombinant adenoviruses ACNTK,AANTK and AANCAT. To construct recombinant adenoviruses from theplasmids described in FIG. 11, 4 parts (20 μg) of either plasmid pACNTK,pAANTK, or pAANCAT were linearized with Eco R1 and cotransfected with 1part (5 μg) of the large fragment of Cla 1 digested recombinantadenovirus (rACβ-gal) containing an E3 region deletion (Wills et al.,1994). In the resulting viruses, the Ad 5 nucleotides 360-4021 arereplaced by either the CMV promoter and tripartite leader cDNA (TPL) orthe α-fetoprotein enhancer and promoter (AFP) driving expression of theHSV-1 or CAT gene as indicated. The resulting recombinant adenovirusesare designated ACNTK, AANTK, and AANCAT respectively.

[0028]FIG. 13 shows promoter specificity of CAT expression in therecombinant adenoviral vectors. Two (2)×10⁶ of the designated cell lineswere infected at MOIs=30 or 100 of the recombinant adenovirus AANCAT asindicated or left uninfected (UN). Hep G2 and Hep 3B cells expressα-fetoprotein whereas the other cell lines do not. After three days, thecells were harvested, extract volumes were adjusted for equal totalprotein concentrations, and CAT activity was measured as described inMethods section, below. An equal number of uninfected cells served asindividual controls for background CAT activity, while ¹⁴C labelledchloramphenicol (¹⁴C-only) and extract from a stable cell line (B21)expressing CAT activity served as negative and positive controlsrespectively. Percent conversion of acetyl CoA is indicated,demonstrating that CAT expression is limited to those cells expressingα-fetoprotein.

[0029]FIG. 14 shows the effects of TK/GCV treatment on hepatocellularcarcinoma cell lines and the effects of promoter specificity. Hep-G2(AFP positive) and HLF (AFP negative) cell lines were infected overnightwith ACNTK [-Δ-] AANTK [-▴-], or control ACN [-□-] virus at an infectionmultiplicity of 30 and subsequently treated with a single dose ofganciclovir at the indicated concentrations. Cell proliferation wasassessed by adding ³H-thymidine to the cells approximately 18 hoursprior to harvest. ³H-thymidine incorporation into cellular nucleic acidwas measured 72 hours after infection (Top Count, Packard and expressedas a percent (mean+/−S.D.) of untreated control. The results show anon-selective dose dependent inhibition of proliferation with the CMVdriven construct, while AFP driven TK selectively inhibits Hep-G2.

[0030]FIG. 15 shows cytotoxicity of ACNTK plus ganciclovir in HCC. HLFcells were infected at an MOI of 30 with either ACNTK [--] or thecontrol virus ACN [-□-] and treated with ganciclovir at the indicateddoses. Seventy-two (72) hours after ganciclovir treatment, the amount oflactate dehydrogenase (LDH) released into the cell supernatant weremeasured calorimetrically and plotted (mean+/−SEM) versus ganciclovirconcentration for the two virus treated groups.

[0031]FIGS. 16A and 16B show the effect of ACNTK plus ganciclovir onestablished hepatocellular carcinoma (HCC) tumors in nude mice. One(1)×10⁷ Hep 3B cells were injected subcutaneously into the flank offemale nude mice and allowed to grow for 27 days. Mice then receivedintratumoral and peritumoral injections of either the ACNTK [--] orcontrol ACN [-□-] virus (1×10⁹ iu in 100 μl volume) every other day fora total of three doses (indicated by arrows). Injections of ganciclovir(100 mg/kg ip) began 24 hours after the initial virus dose and continuedfor a total of 10 days. In FIG. 6A, tumor sizes are plotted for eachvirus versus days post infection (mean+/−SEM). In FIG. 6B, body weightfor each virus-treated animal group is plotted as the mean+/−SEM versusdays post infection.

DETAILED DESCRIPTION OF THE INVENTION

[0032] To reduce the frequency of contamination with wild-typeadenovirus, it is desirable to improve either the virus or the cell lineto reduce the probability of recombination. For example, an adenovirusfrom a group with low homology to the group C viruses could be used toengineer recombinant viruses with little propensity for recombinationwith the Ad5 sequences in 293 cells. However, an alternative, easiermeans of reducing the recombination between viral and cellular sequencesis to increase the size of the deletion in the recombinant virus andthereby reduce the extent of shared sequence between it and the Ad5genes in the 293 cells.

[0033] Deletions which extend past 3.5 kb from the 5′ end of theadenoviral genome affect the gene for adenoviral protein IX and have notbeen considered desirable in adenoviral vectors (see below).

[0034] The protein IX gene of the adenoviruses encodes a minor componentof the outer adenoviral capsid which stabilizes the group-of-nine hexonswhich compose the majority of the viral capsid (Stewart (1993)). Basedupon study of adenovirus deletion mutants, protein IX initially wasthought to be a non-essential component of the adenovirus, although itsabsence was associated with greater heat lability than observed withwild-type virus (Colby and Shenk (1981)). More recently it wasdiscovered that protein IX is essential for packaging full length viralDNA into capsids and that in the absence of protein IX, only genomes atleast 1 kb smaller than wild-type could be propagated as recombinantviruses (Ghosh-Choudhury et al. (1987)). Given this packaginglimitation, protein IX deletions deliberately have not been consideredin the design of adenoviral vectors.

[0035] In this application, reference is made to standard textbooks ofmolecular biology that contain definitions, methods and means forcarrying out basic techniques, encompassed by the present invention. Seefor example, Sambrook et al. (1989) and the various references citedtherein. This reference and the cited publications are expresslyincorporated by reference into this disclosure.

[0036] Contrary to what has been known in the art, this invention claimsthe use of recombinant adenoviruses bearing deletions of the protein IXgene as a means of reducing the risk of wild-type adenoviruscontamination in virus preparations for use in diagnostic andtherapeutic applications such as gene therapy. As used herein, the term“recombinant” is intended to mean a progeny formed as the result ofgenetic engineering. These deletions can remove an additional 500 to 700base pairs of DNA sequence that is present in conventional E1 deletedviruses (smaller, less desirable, deletions of portions of the pIX geneare possible and are included within the scope of this invention) and isavailable for recombination with the Ad5 sequences integrated in 293cells. Recombinant adenoviruses based on any group C virus, serotype 1,2, 5 and 6, are included in this invention. Also encompassed by thisinvention is a hybrid Ad2/Ad5 based recombinant virus expressing thehuman p53 cDNA from the adenovirus type 2 major late promoter. Thisconstruct was assembled as shown in FIG. 1. The resultant virus bears a5′ deletion of adenoviral sequences extending from about nucleotide 357to 4020 and eliminates the E1a and E1b genes as well as the entireprotein IX coding sequence, leaving the polyadenylation site shared bythe E1b and protein IX genes intact for use in terminating transcriptionof any desired gene. A separate embodiment is shown in FIG. 4.Alternatively, the deletion can be extended an additional 30 to 40 basepairs without affecting the adjacent gene for protein IVa2, although inthat case an exogenous polyadenylation signal is provided to terminatetranscription of genes inserted into the recombinant virus. The initialvirus constructed with this deletion is easily propagated in 293 cellswith no evidence of wild-type viral contamination and directs robust p53expression from the transcriptional unit inserted at the site of thedeletion.

[0037] The insert capacity of recombinant viruses bearing the protein IXdeletion described above is approximately 2.6 kb. This is sufficient formany genes including the p53 cDNA. Insert capacity can be increased byintroducing other deletions into the adenoviral backbone, for example,deletions within early regions 3 or 4 (for review see: Graham and Prevec(1991)). For example, the use of an adenoviral backbone containing a 1.9kb deletion of non-essential sequence within early region 3. With thisadditional deletion, the insert capacity of the vector is increased toapproximately 4.5 kb, large enough for many larger cDNAs, including thatof the retinoblastoma tumor suppressor gene.

[0038] A recombinant adenovirus expression vector characterized by thepartial or total deletion of the adenoviral protein IX DNA and having agene encoding a foreign protein, or a functional fragment or mutantthereof is provided by this invention. These vectors are useful for thesafe recombinant production of diagnostic and therapeutic polypeptidesand proteins, and more importantly, for the introduction of genes ingene therapy. Thus, for example, the adenoviral vector of this inventioncan contain a foreign gene for the expression of a protein effective inregulating the cell cycle, such as p53, Rb, or mitosin, or in inducingcell death, such as the conditional suicide gene thymidine kinase. (Thelatter must be used in conjunction with a thymidine kinase metabolite inorder to be effective). Any expression cassette can be used in thevectors of this invention. An “expression cassette” means a DNA moleculehaving a transcription promoter/enhancer such as the CMV promotorenhancer, etc., a foreign gene, and in some embodiments defined below, apolyadentlyation signal. As used herein, the term “foreign gene” isintended to mean a DNA molecule not present in the exact orientation andposition as the counterpart DNA molecule found in wild-type adenovirus.The foreign gene is a DNA molecule up to 4.5 kilobases. “Expressionvector” means a vector that results in the expression of inserted DNAsequences when propagated in a suitable host cell, i.e., the protein orpolypeptide coded for by the DNA is synthesized by the host's system.The recombinant adenovirus expression vector can contain part of thegene encoding adenovirus protein IX, provided that biologically activeprotein IX or fragment thereof is not produced. Example of this vectorare an expression vector having the restriction enzyme map of FIG. 1 or4.

[0039] Inducible promoters also can be used in the adenoviral vector ofthis invention. These promoters will initiate transcription only in thepresence of an additional molecule. Examples of inducible promotersinclude those obtainable from a β-interferon gene, a heat shock gene, ametallothionine gene or those obtainable from steroid hormone-responsivegenes. Tissue specific expression has been well characterized in thefield of gene expression and tissue specific and inducible promoterssuch as these are very well known in the art. These genes are used toregulate the expression of the foreign gene after it has been introducedinto the target cell.

[0040] Also provided by this invention is a recombinant adenovirusexpression vector, as described above, having less extensive deletionsof the protein IX gene sequence extending from 3500 bp from the 5′ viraltermini to approximately 4000 bp, in one embodiment. In a separateembodiment, the recombinant adenovirus expression vector can have afurther deletion of a non-essential DNA sequence in adenovirus earlyregion 3 and/or 4 and/or deletion of the DNA sequences designatedadenovirus E1a and E1b. In this embodiment, foreign gene is a DNAmolecule of a size up to 4.5 kilobases.

[0041] A further embodiment has a deletion of up to forty nucleotidespositioned 3′ to the E1a and E1b deletion and pIX and a foreign DNAmolecule encoding a polyadenylation signal inserted into the recombinantvector in a position relative to the foreign gene to regulate theexpression of the foreign gene.

[0042] For the purposes of this invention, the recombinant adenovirusexpression vector can be derived from wild-type group adenovirus,serotype 1, 2, 5 or 6.

[0043] In one embodiment, the recombinant adenovirus expression vectorhas a foreign gene coding for a functional tumor suppressor protein, ora biologically active fragment thereof. As used herein, the term“functional” as it relates to a tumor suppressor gene, refers to tumorsuppressor genes that encode tumor suppressor proteins that effectivelyinhibit a cell from behaving as a tumor cell. Functional genes caninclude, for instance, wild type of normal genes and modifications ofnormal genes that retains its ability to encode effective tumorsuppressor proteins and other anti-tumor genes such as a conditionalsuicide protein or a toxin.

[0044] Similarly, “non-functional” as used herein is synonymous with“inactivated.” Non-functional or defective genes can be caused by avariety of events, including for example point mutations, deletions,methylation and others known to those skilled in the art.

[0045] As used herein, an “active fragment” of a gene includes smallerportions of the gene that retain the ability to encode proteins havingtumor suppressing activity. p56^(RB), described more fully below, is butone example of an active fragment of a functional tumor suppressor gene.Modifications of tumor suppressor genes are also contemplated within themeaning of an active fragment, such as additions, deletions orsubstitutions, as long as the functional activity of the unmodified geneis retained.

[0046] Another example of a tumor suppressor gene is retinoblastoma(RB). The complete RB cDNA nucleotide sequences and predicted amino acidsequences of the resulting RB protein (designated p110^(RB)) are shownin Lee et al. (1987) and in FIG. 3. Also useful to expressretinoblastoma tumor suppressor protein is a DNA molecule encoding theamino acid sequence shown in FIG. 2 or having the DNA sequence shown inFIG. 3. A truncated version of p110^(RB), called p56^(RB) also isuseful. For the sequence of p56^(RB), see Huang et al. (1991).Additional tumor suppressor genes can be used in the vectors of thisinvention. For illustration purposes only, these can be p16 protein(Kamb et al. (1994)), p21 protein, Wilm's tumor WT1 protein, mitosin,h-NUC, or colon carcinoma DCC protein. Mitosin is described in X. Zhuand W-H Lee, U.S. application Ser. No. 08/141,239, filed Oct. 22, 1993,and a subsequent continuation-in-part by the same inventors, attorneydocket number P-CJ 1191, filed Oct. 24, 1994, both of which are hereinincorporated by reference. Similarly, h-NUC is described by W-H Lee andP-L Chen, U.S. application Ser. No. 08/170,586, filed Dec. 20, 1993,herein incorporated by reference.

[0047] As is known to those of skill in the art, the term “protein”means a linear polymer of amino acids joined in a specific sequence bypeptide bonds. As used herein, the term “amino acid” refers to eitherthe D or L stereoisomer form of the amino acid, unless otherwisespecifically designated. Also encompassed within the scope of thisinvention are equivalent proteins or equivalent peptides, e.g., havingthe biological activity of purified wild type tumor suppressor protein.“Equivalent proteins” and “equivalent polypeptides” refer to compoundsthat depart from the linear sequence of the naturally occurring proteinsor polypeptides, but which have amino acid substitutions that do notchange its biologically activity. These equivalents can differ from thenative sequences by the replacement of one or more amino acids withrelated amino acids, for example, similarly charged amino acids, or thesubstitution or modification of side chains or functional groups.

[0048] Also encompassed within the definition of a functional tumorsuppressor protein is any protein whose presence reduces thetumorigenicity, malignancy or hyperproliferative phenotype of the hostcell. Examples of tumor suppressor proteins within this definitioninclude, but are not limited to p110^(RB), p56^(RB), mitosin, h-NUC andp53. “Tumorigenicity” is intended to mean having the ability to formtumors or capable of causing tumor formation and is synonymous withneoplastic growth. “Malignancy” is intended to describe a tumorigeniccell having the ability to metastasize and endanger the life of the hostorganism. “Hyperproliferative phenotype” is intended to describe a cellgrowing and dividing at a rate beyond the normal limitations of growthfor that cell type. “Neoplastic” also is intended to include cellslacking endogenous functional tumor suppressor protein or the inabilityof the cell to express endogenous nucleic acid encoding a functionaltumor suppressor protein.

[0049] An example of a vector of this invention is a recombinantadenovirus expression vector having a foreign gene coding for p53protein or an active fragment thereof is provided by this invention. Thecoding sequence of the p53 gene is set forth below in Table I. TABLE 1                                                                  50V*SHR  PGSR*  LLGSG  DTLRS  GWERA  FHDGD  TLPWI  GSQTA  FRVTA  MEEPQ                                                                 100SDPSV  EPPLS  QETFS  DLWKL  LPENN  VLSPL  PSQAM  DDLML  SPDDI  EQWFT                                                                 150EDPGP  DEAPR  MPEAA  PPVAP  APAAP  TPAAP  APAPS  WPLSS  SVPSQ  KTYQG                                                                 200SYGPR  LGFLH  SGTAK  SVTCT  YSPAL  NKMFC  QLAKT  CPVQL  WVDST  PPPGT                                                                 250RVRAM  AIYKQ  SQHMT  EVVRR  CPHHE  RCSDS  DGLAP  PQHLI  RVEGN  LRVEY                                                                 300LDDRN  TFRHS  VVVPY  EPPEV  GSDCT  TIHYN  YMCNS  SCMGG  MNRRP  ILTII                                                                 350TLEDS  SGNLL  GRNSF  EVRVC  ACPGR  DRRTE  EENLR  KKGEP  HHELP  PGSTK                                                                 400RALPN  NTSSS  PQPKK  KPLDG  EYFTL  QIRGR  ERFEM  FRELN  EALEL  KDAQAGKEPG  GSRAH  SSHLK  SKKGQ  STSRH  KKLMF  KTEGP  DSD*

[0050] Any of the expression vectors described herein are useful ascompositions for diagnosis or therapy. The vectors can be used forscreening which of many tumor suppressor genes would be useful in genetherapy. For example, a sample of cells suspected of being neoplasticcan be removed from a subject and mammal. The cells can then becontacted, under suitable conditions and with an effective amount of arecombinant vector of this invention having inserted therein a foreigngene encoding one of several functional tumor suppressor genes. Whetherthe introduction of this gene will reverse the malignant phenotype canbe measured by colony formation in soft agar or tumor formation in nudemice. If the malignant phenotype is reversed, then that foreign gene isdetermined to be a positive candidate for successful gene therapy forthe subject or mammal. When used pharmaceutically, they can be combinedwith one or more pharmaceutically acceptable carriers. Pharmaceuticallyacceptable carriers are well known in the art and include aqueoussolutions such as physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, vegetable oils (eg., olive oil) orinjectable organic esters. A pharmaceutically acceptable carrier can beused to administer the instant compositions to a cell in vitro or to asubject in vivo.

[0051] A pharmaceutically acceptable carrier can contain aphysiologically acceptable compound that acts, for example, to stabilizethe composition or to increase or decrease the absorption of the agent.A physiologically acceptable compound can include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. Other physiologicallyacceptable compounds include wetting agents, emulsifying agents,dispersing agents or preservatives, which are particularly useful forpreventing the growth or action of microorganisms. Various preservativesare well known and include, for example, phenol and ascorbic acid. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the polypeptideand on the particular physio-chemical characteristics of the specificpolypeptide. For example, a physiologically acceptable compound such asaluminum monosterate or gelatin is particularly useful as a delayingagent, which prolongs the rate of absorption of a pharmaceuticalcomposition administered to a subject. Further examples of carriers,stabilizers or adjutants can be found in Martin, Remington's Pharm.Sci., 15th Ed. (Mack Publ. Co., Easton, 1975), incorporated herein byreference. The pharmaceutical composition also can be incorporated, ifdesired, into liposomes, microspheres or other polymer matrices(Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla.1984), which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer.

[0052] As used herein, “pharmaceutical composition” refers to any of thecompositions of matte described herein in combination with one or moreof the above pharmaceutically acceptable carriers. The compositions canthen be administered therapeutically or prophylactically. They can becontacted with the host cell in vivo, ex vivo, or in vitro, in aneffective amount. In vitro and ex vivo means of contacting host cellsare provided below. When practiced in vivo, methods of administering apharmaceutical containing the vector of this invention, are well knownin the art and include but are not limited to, administration orally,intra-tumorally, intravenously, intramuscularly or intraperitoneal.Administration can be effected continuously or intermittently and willvary with the subject and the condition to be treated, e.g., as is thecase with other therapeutic compositions (Landmann et al. (1992);Aulitzky et al. (1991); Lantz et al. (1990); Supersaxo et al. (1988);Demetri et al. (1989); and LeMaistre et al. (1991)).

[0053] Further provided by this invention is a transformed procaryoticor eucaryotic host cell, for example an animal cell or mammalian cell,having inserted a recombinant adenovirus expression vector describedabove. Suitable procaryotic cells include but are not limited tobacterial cells such as E. coli cells. Methods of transforming hostcells with retroviral vectors are known in the art, see Sambrook et al.(1989) and include, but are not limited to transfection,electroporation, and microinjection.

[0054] As used throughout this application, the term animal is intendedto be synonymous with mammal and is to include, but not be limited tobovine, porcine, feline, simian, canine, equine, murine, rat or human.Additional host cells include but are not limited to any neoplastic ortumor cell, such as osteosarcoma, ovarian carcinoma, breast carcinoma,melanoma, hepatocarcinoma, lung cancer, brain cancer, colorectal cancer,hematopoietic cell, prostate cancer, cervical carcinoma, retinoblastoma,esophageal carcinoma, bladder cancer, neuroblastoma, or renal cancer.

[0055] Additionally, any eucaryotic cell line capable of expressing E1aand E1b or E1a, E1b and pIX is a suitable host for this vector. In oneembodiment, a suitable eucaryotic host cell is the 293 cell lineavailable from the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., U.S.A. 20231.

[0056] Any of the transformed host cells described herein are useful ascompositions for diagnosis or therapy. When used pharmaceutically, theycan be combined with various pharmaceutically acceptable carriers.Suitable pharmaceutically acceptable carriers are well known to those ofskill in the art and, for example, are described above. The compositionscan then be administered therapeutically or prophylactically, ineffective amounts, described in more detail below.

[0057] A method of transforming a host cell also is provided by thisinvention. This method provides contacting a host cell, i.e., aprocaryotic or eucaryotic host cell, with any of the expression vectorsdescribed herein and under suitable conditions. Host cells transformedby this method also are claimed within the scope of this invention. Thecontacting can be effected in vitro, in vivo, or ex vivo, using methodswell known in the art (Sambrook et al. (1989)) and using effectiveamounts of the expression vectors. Also provided in this invention is amethod of producing a recombinant protein or polypeptide by growing thetransformed host cell under suitable conditions favoring thetranscription and translation of the inserted foreign gene. Methods ofrecombinant expression in a variety of host cells, such as mammalian,yeast, insect or bacterial cells, are widely known, including thosedescribed in Sambrook et al., supra. The translated foreign gene canthen be isolated by convention means, such as column purification orpurification using an anti-protein antibody. The isolated protein orpolypeptide also is intended within the scope of this invention. As usedherein, purified or isolated mean substantially free of native proteinsor nucleic acids normally associated with the protein or polypeptide inthe native or host cell environment.

[0058] Also provided by this invention are non-human animals havinginserted therein the expression vectors or transformed host cells ofthis invention. These “transgenic” animals are made using methods wellknown to those of skill in the art, for example as described in U.S.Pat. No. 5,175,384 or by conventional ex vivo therapy techniques, asdescribed in Culver et al. (1991).

[0059] As shown in detail below, the recombinant adenoviruses expressinga tumor suppressor wild-type p53, as described above, can efficientlyinhibit DNA synthesis and suppress the growth of a broad range of humantumor cell types, including clinical targets. Furthermore, recombinantadenoviruses can express tumor suppression genes such as p53 in an invivo established tumor without relying on direct injection into thetumor or prior ex vivo treatment of the cancer cells. The p53 expressedis functional and effectively suppresses tumor growth in vivo andsignificantly increases survival time in a nude mouse model of humanlung cancer.

[0060] Thus, the vectors of this invention are particularly suited forgene therapy. Accordingly, methods of gene therapy utilizing thesevectors are within the scope of this invention. The vector is purifiedand then an effective amount is administered in vivo or ex vivo into thesubject. Methods of gene therapy are well known in the art, see, forexample, Larrick, J. W. and Burck, K. L. (1991) and Kreigler, M. (1990).“Subject” means any animal, mammal, rat, murine, bovine, porcine,equine, canine, feline or human patient. When the foreign gene codes fora tumor suppressor gene or other anti-tumor protein, the vector isuseful to treat or reduce hyperproliferative cells in a subject, toinhibit tumor proliferation in a subject or to ameliorate a particularrelated pathology. Pathologic hyperproliferative cells arecharacteristic of the following disease states, thyroidhyperplasia—Grave's Disease, psoriasis, benign prostatic hypertrophy,Li-Fraumeni syndrome including breast cancer, sarcomas and otherneoplasms, bladder cancer, colon cancer, lung cancer, various leukemiasand lymphomas. Examples of non-pathologic hyperproliferative cells arefound, for instance, in mammary ductal epithelial cells duringdevelopment of lactation and also in cells associated with wound repair.Pathologic hyperproliferative cells characteristically exhibit loss ofcontact inhibition and a decline in their ability to selectively adherewhich implies a change in the surface properties of the cell and afurther breakdown in intercellular communication. These changes includestimulation to divide and the ability to secrete proteolytic enzymes.

[0061] Moreover, the present invention relates to a method for depletinga suitable sample of pathologic mammalian hyperproliferative cellscontaminating hematopoietic precursors during bone marrow reconstitutionvia the introduction of a wild type tumor suppressor gene into the cellpreparation using the vector of this invention (whether derived fromautologous peripheral blood or bone marrow). As used herein, a “suitablesample” is defined as a heterogeneous cell preparation obtained from apatient, e.g., a mixed population of cells containing bothphenotypically normal and pathogenic cells. “Administer” includes, butis not limited to introducing into the cell or subject intravenously, bydirect injection into the tumor, by intra-tumoral injection, byintraperitoneal administration, by aerosol administration to the lung ortopically. Such administration can be combined with apharmaceutically-accepted carrier, described above.

[0062] The term “reduced tumorigenicity” is intended to mean tumor cellsthat have been converted into less tumorigenic or non-tumorigenic cells.Cells with reduced tumorigenicity either form no tumors in vivo or havean extended lag time of weeks to months before the appearance of in vivotumor growth and/or slower growing three dimensional tumor mass comparedto tumors having fully inactivated or non-functional tumor suppressorgene.

[0063] As used herein, the term “effective amount” is intended to meanthe amount of vector or anti-cancer protein which achieves a positiveoutcome on controlling cell proliferation. For example, one dosecontains from about 10⁸ to about 10¹³ infectious units. A typical courseof treatment would be one such dose a day over a period of five days. Aneffective amount will vary on the pathology or condition to be treated,by the patient and his status, and other factors well known to those ofskill in the art. Effective amounts are easily determined by those ofskill in the art.

[0064] Also within the scope of this invention is a method ofameliorating a pathology characterized by hyperproliferative cells orgenetic defect in a subject by administering to the subject an effectiveamount of a vector described above containing a foreign gene encoding agene product having the ability to ameliorate the pathology, undersuitable conditions. As used herein, the term “genetic defect” means anydisease or abnormality that results from inherited factors, such assickle cell anemia or Tay-Sachs disease.

[0065] This invention also provides a method for reducing theproliferation of tumor cells in a subject by introducing into the tumormass an effective amount of an adenoviral expression vector containingan anti-tumor gene other than a tumor suppressor gene. The anti-tumorgene can encode, for example, thymidine kinase (TK). The subject is thenadministered an effective amount of a therapeutic agent, which in thepresence of the anti-tumor gene is toxic to the cell. In the specificcase of thymidine kinase, the therapeutic agent is a thymidine kinasemetabolite such as ganciclovir (GCV), 6-methoxypurine arabinonucleoside(araM), or a functional equivalent thereof. Both the thymidine kinasegene and the thymidine kinase metabolite must be used concurrently to betoxic to the host cell. However, in its presence, GCV is phosphorylatedand becomes a potent inhibitor of DNA synthesis whereas araM getsconverted to the cytotoxic anabolite araATP. Other anti-tumor genes canbe used as well in combination with the corresponding therapeutic agentto reduce the proliferation of tumor cells. Such other gene andtherapeutic agent combinations are known by one skilled in the art.Another example would be the vector of this invention expressing theenzyme cytosine deaminase. Such vector would be used in conjunction withadministration of the drug 5-fluorouracil (Austin and Huber, 1993), orthe recently described E. Coli Deo Δ gene in combination with6-methyl-purine-2′-deosribonucleoside (Sorscher et al 1994).

[0066] As with the use of the tumor suppressor genes describedpreviously, the use of other anti-tumor genes, either alone or incombination with the appropriate therapeutic agent provides a treatmentfor the uncontrolled cell growth or proliferation characteristic oftumors and malignancies. Thus, this invention provides a therapy to stopthe uncontrolled cellular growth in the patient thereby alleviating thesymptoms of the disease or cachexia present in the patient. The effectof this treatment includes, but is not limited to, prolonged survivaltime of the patient, reduction in tumor mass or burden, apoptosis oftumor cells or the reduction of the number of circulating tumor cells.Means of quantifying the beneficial effects of this therapy are wellknown to those of skill in the art.

[0067] The invention provides a recombinant adenovirus expression vectorcharacterized by the partial or total deletion of the adenoviral proteinIX DNA and having a foreign gene encoding a foreign protein, wherein theforeign protein is a suicide gene or functional equivalent thereof. Theanti-cancer gene TK, described above, is an example of a suicide genebecause when expressed, the gene product is, or can be made to be lethalto the cell. For TK, lethality is induced in the presence of GCV. The TKgene is derived from herpes simplex virus by methods well known to thoseof skill in the art. The plasmid pMLBKTK in E. coli HB101 (from ATCC#39369) is a source of the herpes simplex virus (HSV-1) thymidine kinase(TK) gene for use in this invention. However, many other sources existas well.

[0068] The TK gene can be introduced into the tumor mass by combiningthe adenoviral expression vector with a suitable pharmaceuticallyacceptable carrier. Introduction can be accomplished by, for example,direct injection of the recombinant adenovirus into the tumor mass. Forthe specific case of a cancer such as hepatocellular carcinoma (HCC),direct injection into the hepatic artery can be used for deliverybecause most HCCs derive their circulation from this artery. To controlproliferation of the tumor, cell death is induced by treating thepatients with a TK metabolite such as ganciclovir to achieve reductionof tumor mass. The TK metabolite can be administered, for example,systemically, by local innoculation into the tumor or in the specificcase of HCC, by injection into the hepatic artery. The TK metabolite ispreferably administered at least once daily but can be increased ordecreased according to the need. The TK metabolite can be administeredsimultaneous or subsequent to the administration of the TK containingvector. Those skilled in the art know or can determine the dose andduration which is therapeutically effective.

[0069] A method of tumor-specific delivery of a tumor suppressor gene isaccomplished by contacting target tissue in an animal with an effectiveamount of the recombinant adenoviral expression vector of thisinvention. The gene is intended to code for an anti-tumor agent, such asa functional tumor suppressor gene or suicide gene. “Contacting” isintended to encompass any delivery method for the efficient transfer ofthe vector, such as intra-tumoral injection.

[0070] The use of the adenoviral vector of this invention to preparemedicaments for the treatment of a disease or for therapy is furtherprovided by this invention.

[0071] The following examples are intended to illustrate, not limit thescope of this invention.

EXPERIMENT NO. I

[0072] Plasmid pAd/MLP/p53/E1b—was used as the starting material forthese manipulations. This plasmid is based on the pBR322 derivative pML2(pBR322 deleted for base pairs 1140 to 2490) and contains adenovirustype 5 sequences extending from base pair 1 to base pair 5788 exceptthat it is deleted for adenovirus type 5 base pairs 357 to 3327. At thesite of the Ad5 357/3327 deletion a transcriptional unit is insertedwhich is comprised of the adenovirus type 2 major late promoter, theadenovirus type 2 tripartite leader cDNA and the human p53 cDNA. It is atypical E1 replacement vector deleted for the Ad5 E1a and E1b genes butcontaining the Ad5 protein IX gene (for review of Adenovirus vectorssee: Graham and Prevec (1992)). Ad2 DNA was obtained from Gibco BRL.Restriction endonucleases and T4 DNA ligase were obtained from NewEngland Biolabs. E. coli DH5α competent cells were purchased from GibcoBRL and 293 cells were obtained from the American Type CultureCollection (ATCC). Prep-A-Gene DNA purification resin was obtained fromBioRad. LB broth bacterial growth medium was obtained from Difco. QiagenDNA purification columns were obtained from Qiagen, Inc. Ad5 dl327 wasobtained from R. J. Schneider, NYU. The MBS DNA transfection kit waspurchased from Stratagene.

[0073] One (1) μg pAd/MLP/p53/E1b—was digested with 20 units each ofrestriction enzymes Ecl 136II and NgoMI according to the manufacturer'srecommendations. Five (5) μg Ad2 DNA was digested with 20 units each ofrestriction endonucleases DraI and NgoMI according to the manufacturer'srecommendations. The restriction digestions were loaded into separatelanes of a 0.8% agarose gel and electrophoresed at 100 volts for 2hours. The 4268 bp restriction fragment from the Pad/MLP/p53/E1b—sampleand the 6437 bp fragment from the Ad2 sample were isolated from the gelusing Prep-A-Gene DNA extraction resin according to the manufacturer'sspecifications. The restriction fragments were mixed and treated with T4DNA ligase in a total volume of 50 μl at 16° C. for 16 hours accordingto the manufacturer's recommendations. Following ligation 5 μl of thereaction was used to transform E. coli DH5α cells to ampicillinresistance following the manufacturer's procedure. Six bacterialcolonies resulting from this procedure were used to inoculate separate 2ml cultures of LB growth medium and incubated overnight at 37° C. withshaking. DNA was prepared from each bacterial culture using standardprocedures (Sambrook et al. (1989)). One fourth of the plasmid DNA fromeach isolate was digested with 20 units of restriction endonuclease XhoIto screen for the correct recombinant containing XhoI restrictionfragments of 3627, 3167, 2466 and 1445 base pairs. Five of six screenedisolates contained the correct plasmid. One of these was then used. toinoculate a 1 liter culture of LB medium for isolation of largequantities of plasmid DNA. Following overnight incubation plasmid DNAwas isolated from the 1 liter culture using Qiagen DNA purificationcolumns according to the manufacturer's recommendations. The resultingplasmid was designated Pad/MLP/p53/PIX—Samples of this plasmid weredeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., U.S.A., 12301, on Oct. 22, 1993. The deposit wasmade under the provisions of the Budapest Treaty on the InternationalDeposit of Microorganisms for the Purpose of Patent Procedure. Thedeposit was accorded ATCC Accession No. 75576.

[0074] To construct a recombinant adenovirus, 10 μg Pad/MLP/p53/PIX—weretreated with 40 units of restriction endonuclease EcoRI to linearize theplasmid. Adenovirus type 5 dl327 DNA (Thimmappaya (1982)) was digestedwith restriction endonuclease ClaI and the large fragment (approximately33 kilobase pairs) was purified by sucrose gradient centrifugation. Ten(10) μg of EcoRI treated Pad/MLP/p53/E1b and 2.5 μg of ClaI treated Ad5dl327 were mixed and used to transfect approximately 10⁶ 293 cells usingthe MBS mammalian transfection kit as recommended by the supplier. Eight(8) days following the transfection the 293 cells were split 1 to 3 intofresh media and two days following this adenovirus induced cytopathiceffect became evident on the transfected cells. At 13 dayspost-transfection DNA was prepared from the infected cells usingstandard procedures (Graham and Prevec (1991)) and analyzed byrestriction digestion with restriction endonuclease XhoI. Virus directedexpression of p53 was verified following infection of SaoS2 osteosarcomacells with viral lysate and immunoblotting with an anti-p53 monoclonalantibody designated 1801 (Novocasta Lab. Ltd., U.K.).

EXPERIMENT NO. II

[0075] Materials and Methods

[0076] Cell Lines

[0077] Recombinant adenoviruses were grown and propagated in the humanembryonal kidney cell line 293 (ATCC CRL 1573) maintained in DME mediumcontaining 10% defined, supplemented calf serum (Hyclone). Saos-2 cellswere maintained in Kaighn's media supplemented with 15% fetal calfserum. HeLa and Hep 3B cells were maintained in DME medium supplementedwith 10% fetal calf serum. All other cell lines were grown in Kaighn'smedia supplemented with 10% fetal calf serum. Saos-2 cells were kindlyprovided by Dr. Eric Stanbridge. All other cell lines were obtained fromATCC.

[0078] Construction of Recombinant Adenoviruses

[0079] To construct the Ad5/p53 viruses, a 1.4 kb HindIII-SmaI fragmentcontaining the full length cDNA for p53 (Table I) was isolated frompGEM1-p53-B-T (kindly supplied by Dr. Wen Hwa Lee) and inserted into themultiple cloning site of the expression vector pSP72 (Promega) usingstandard cloning procedures (Sambrook et al. (1989)). The p53 insert wasrecovered from this vector following digestion with XhoI-BglII and gelelectrophoresis. The p53 coding sequence was then inserted into eitherpNL3C or pNL3CMV adenovirus gene transfer vectors (kindly provided byDr. Robert Schneider) which contain the Ad5 5′ inverted terminal repeatand viral packaging signals and the E1a enhancer upstream of either theAd2 major late promoter (MLP) or the human cytomegalovirus immediateearly gene promoter (CMV), followed by the tripartite leader CDNA and Ad5 sequence 3325-5525 bp in a PML2 background. These new constructsreplace the E1 region (bp 360-3325) of Ad5 with p53 driven by either theAd2 MLP (A/M/53) or the human CMV promoter (A/C/53), both followed bythe tripartite leader cDNA (see FIG. 4). The p53 inserts use theremaining downstream E1b polyadenylation site. Additional MLP and CMVdriven p53 recombinants (A/M/N/53, A/C/N/53) were generated which had afurther 705 nucleotide deletion of Ad 5 sequence to remove the proteinIX (PIX) coding region. As a control, a recombinant adenovirus wasgenerated from the parental PNL3C plasmid without a p53 insert (A/M). Asecond control consisted of a recombinant adenovirus encoding thebeta-galactosidase gene under the control of the CMV promoter(A/C/β-gal). The plasmids were linearized with either Nru I or Eco RIand co-transfected with the large fragment of a Cla I digested Ad 5dl309 or dl327 mutants (Jones and Shenk (1979)) using a Ca/PO₄transfection kit (Stratagene). Viral plaques were isolated andrecombinants identified by both restriction digest analysis and PCRusing recombinant specific primers against the tripartite leader CDNAsequence with downstream p53 CDNA sequence. Recombinant virus wasfurther purified by limiting dilution, and virus particles were purifiedand titered by standard methods (Graham and van der Erb (1973); Grahamand Prevec (1991)).

[0080] p53 Protein Detection

[0081] Saos-2 or Hep 3B cells (5×10⁵) were infected with the indicatedrecombinant adenoviruses for a period of 24 hours at increasingmultiplicities of infection (MOI) of plaque forming units of virus/cell.Cells were then washed once with PBS and harvested in lysis buffer (50mM Tris-Hcl Ph 7.5, 250 Mm NaCl, 0.1% NP40, 50 mM NaF, 5 mM EDTA, 10ug/ml aprotinin, 10 ug/ml leupeptin, and 1 mM PMSF). Cellular proteins(approximately 30 μg) were separated by 10% SDS-PAGE and transferred tonitrocellulose. Membranes were incubated with α-p53 antibody PAb 1801(Novocastro) followed by sheep anti-mouse IgG conjugated withhorseradish peroxidase. p53 protein was visualized by chemiluminescence(ECL kit, Amersham) on Kodak XAR-5 film.

[0082] Measurement of DNA Synthesis Rate

[0083] Cells (5×10³/well) were plated in 96-well titer plates (Costar)and allowed to attach overnight (37° C., 7% CO₂). Cells were theninfected for 24 hours with purified recombinant virus particles at MOIsranging from 0.3 to 100 as indicated. Media were changed 24 hours afterinfection, and incubation was continued for a total of 72 hours.³H-thymidine (Amersham, 1 μCi/well) was added 18 hours prior to harvest.Cells were harvested on glass fiber filters and levels of incorporatedradioactivity were measured in a beta scintillation counter.³H-thymidine incorporation was expressed as the mean % (+/−SD) of mediacontrol and plotted versus the MOI.

[0084] Tumorigenicity in Nude Mice

[0085] Approximately 2.4×10⁸ Saos-2 cells, plated in T225 flasks, weretreated with suspension buffer (1% sucrose in PBS) containing eitherA/M/N/53 or A/M purified virus at an MOI of 3 or 30. Following anovernight infection, cells were injected subcutaneously into the leftand right flanks of BALB/c athymic nude mice (4 mice per group). Oneflank was injected with the A/M/N/53 treated cells, while thecontralateral flank was injected with the control A/M treated cells,each mouse serving as its own control. Animals receiving bilateralinjection of buffer treated cells served as additional controls. Tumordimensions (length, width and height) and body weights were thenmeasured twice per week over an 8 week period. Tumor volumes wereestimated for each animal assuming a spherical geometry with radiusequal to one-half the average of the measured tumor dimensions.

[0086] Intra-Tumoral RNA Analysis

[0087] BALB/c athymic nude mice (approximately 5 weeks of age) wereinjected subcutaneously with 1×10⁷ H69 small cell lung carcinoma (SCLC)cells in their right flanks. Tumors were allowed to progress for 32 daysuntil they were approximately 25-50 mm³. Mice received peritumoralinjections of either A/C/53 or A/C/β-gal recombinant adenovirus (2×10⁹plaque forming units (pfu)) into the subcutaneous space beneath thetumor mass. Tumors were excised from the animals 2 and 7 days postadenovirus treatment and rinsed with PBS. Tumor samples werehomogenized, and total RNA was isolated using a TriReagent kit(Molecular Research Center, Inc.). PolyA RNA was isolated using thePolyATract mRNA Isolation System (Promega), and approximately 10 ng ofsample was used for RT-PCR determination of recombinant p53 MRNAexpression (Wang et al. (1989)). Primers were designed to amplifysequence between the adenovirus tripartite leader CDNA and thedownstream p53 CDNA, ensuring that only recombinant, and not endogenousp53 would be amplified.

[0088] p53 Gene Therapy of Established Tumors in Nude Mice

[0089] Approximately 1×10⁷ H69 (SCLC) tumor cells in 2001 μl volumeswere injected subcutaneously into female BALB/c athymic nude mice.Tumors were allowed to develop for 2 weeks, at which point animals wererandomized by tumor size (N=5/group). Peritumoral injections of eitherA/M/N/53 or the control A/M adenovirus (2×10⁹ pfu/injection) or bufferalone (1% sucrose in PBS) were administered twice per week for a totalof 8 doses/group. Tumor dimensions and body weights were measured twiceper week for 7 weeks, and tumor volume was estimated as describedpreviously. Animals were then followed to observe the effect oftreatment on mouse survival.

[0090] Results

[0091] Construction of Recombinant p53-Adenovirus

[0092] p53 adenoviruses were constructed by replacing a portion of theE1a and E1b region of adenovirus Type 5 with p53 CDNA under the controlof either the Ad2 MLP (A/M/53) or CMV (A/C/53) promoter (schematized inFIG. 4). This E1 substitution severely impairs the ability of therecombinant adenoviruses to replicate, restricting their propagation to293 cells which supply Ad 5 E1 gene products in trans (Graham et al.(1977)). After identification of p53 recombinant adenovirus by bothrestriction digest and PCR analysis, the entire p53 CDNA sequence fromone of the recombinant adenoviruses (A/M/53) was sequenced to verifythat it was free of mutations. Following this, purified preparations ofthe p53 recombinants were used to infect HeLa cells to assay for thepresence of phenotypically wild type adenovirus. HeLa cells, which arenon-permissive for replication of E1-deleted adenovirus, were infectedwith 1-4×10⁹ infectious units of recombinant adenovirus, cultured for 3weeks, and observed for the appearance of cytopathic effect (CPE). Usingthis assay, recombinant adenovirus replication or wild typecontamination was not detected, readily evident by the CPE observed incontrol cells infected with wild type adenovirus at a level ofsensitivity of approximately 1 in 10⁹.

[0093] p53 Protein Expression from Recombinant Adenovirus

[0094] To determine if p53 recombinant adenoviruses expressed p53protein, tumor cell lines which do not express endogenous p53 proteinwere infected. The human tumor cell lines Saos-2 (osteosarcoma) and Hep3B (hepatocellular carcinoma) were infected for 24 hours with the p53recombinant adenoviruses A/M/53 or A/C/53 at MOIs ranging 0.1 to 200pfu/cell. Western analysis of lysates prepared from infected cellsdemonstrated a dose-dependent p53 protein expression in both cell types(FIG. 5). Both cell lines expressed higher levels of p53 proteinfollowing infection with A/C/53 than with A/M/53 (FIG. 3). No p53protein was detected in non-infected cells. Levels of endogenouswild-type p53 are normally quite low, and nearly undetectable by Westernanalysis of cell extracts (Bartek et al. (1991)). It is clear howeverthat wild-type p53 protein levels are easily detectable after infectionwith either A/M/53 or A/C/53 at the lower MOIs (FIG. 5), suggesting thateven low doses of p53 recombinant adenoviruses can produce potentiallyefficacious levels of p53.

[0095] p53 Dependent Morphology Changes

[0096] The reintroduction of wild-type p53 into the p53-negativeosteosarcoma cell line, Saos-2, results in a characteristic enlargementand flattening of these normally spindle-shaped cells (Chen et al.(1990)). Subconfluent Saos-2 cells (1×10⁵ cells/10 cm plate) wereinfected at an MOI of 50 with either the A/C/53 or control A/M virus,and incubated at 37° C. for 72 hours until uninfected control plateswere confluent. At this point, the expected morphological change wasevident in the A/C/53 treated plate (FIG. 6, panel C) but not inuninfected (FIG. 6, panel A) or control virus-infected plates (FIG. 6,panel B). This effect was not a function of cell density because acontrol plate initially seeded at lower density retained normalmorphology at 72 hours when its confluence approximated that of theA/C/53 treated plate. Previous results had demonstrated a high level ofp53 protein expression at an MOI of 50 in Saos-2 cells (FIG. 5A), andthese results provided evidence that the p53 protein expressed by theserecombinant adenoviruses was biologically active.

[0097] p53 Inhibition of Cellular DNA Synthesis

[0098] To further test the activity of the p53 recombinant adenoviruses,their ability to inhibit proliferation of human tumor cells was assayedas measured by the uptake of ³H-thymidine. It has previously been shownthat introduction of wild-type p53 into cells which do not expressendogenous wild-type p53 can arrest the cells at the G₁/S transition,leading to inhibition of uptake of labeled thymidine into newlysynthesized DNA (Baker et al. (1990); Mercer et al. (1990); Diller etal. (1990)). A variety of p53-deficient tumor cell lines were infectedwith either A/M/N/53, A/C/N/53 or a non-p53 expressing controlrecombinant adenovirus (A/M). A strong, dose-dependent inhibition of DNAsynthesis by both the A/M/N/53 and A/C/N/53 recombinants in 7 out of the9 different tumor cell lines tested (FIG. 7) was observed. Bothconstructs were able to inhibit DNA synthesis in these human tumorcells, regardless of whether they expressed mutant p53 or failed toexpress p53 protein. It also was found that in this assay, the A/C/N/53construct was consistently more potent than the A/M/N/53. In saos-2(osteosarcoma) and MDA-MB468 (breast cancer) cells, nearly 100%inhibition of DNA synthesis was achieved with the A/C/N/53 construct atan MOI as low as 10. At doses where inhibition by the control adenovirusin only 10-30%, a 50-100% reduction in DNA synthesis using either p53recombinant adenovirus was observed. In contrast, no significantp53-specific effect was observed with either construct as compared tocontrol virus in HEP G2 cells (hepatocarcinoma cell line expressingendogenous wild-type p53, Bressac et al. (1990)), nor in the K562 (p53null) leukemic cell line.

[0099] Tumorigenicity in Nude Mice

[0100] In a more stringent test of function for the p53 recombinantadenoviruses, tumor cells were infected ex vivo and then injected thecells into nude mice to assess the ability of the recombinants tosuppress tumor growth in vivo. Saos-2 cells infected with A/M/N/53 orcontrol A/M virus at a MOI of 3 or 30, were injected into oppositeflanks of nude mice. Tumor sizes were then measured twice a week over an8 week period. At the MOI of 30, no tumor growth was observed in thep53-treated flanks in any of the animals, while the control treatedtumors continued to grow (FIG. 8). The progressive enlargement of thecontrol virus treated tumors were similar to that observed in the buffertreated control animals. A clear difference in tumor growth between thecontrol adenovirus and the p53 recombinant at the MOI of 3, althoughtumors from 2 out of the 4 p53-treated mice did start to show somegrowth after approximately 6 weeks. Thus, the A/M/N/53 recombinantadenovirus is able to mediate p53-specific tumor suppression in an invivo environment.

[0101] In Vivo Expression of Ad/p53

[0102] Although ex vivo treatment of cancer cells and subsequentinjection into animals provided a critical test of tumor suppression, amore clinically relevant experiment is to determine if injected p53recombinant adenovirus could infect and express p53 in establishedtumors in vivo. To address this, H69 (SCLC, p53^(null)) cells wereinjected subcutaneously into nude mice, and tumors were allowed todevelop for 32 days. At this time, a single injection of 2×10⁹ pfu ofeither A/C/53 or A/C/β-gal adenovirus was injected into the peritumoralspace surrounding the tumor. Tumors were then excised at either Day 2 orDay 7 following the adenovirus injection, and polyA RNA was isolatedfrom each tumor. RT-PCR, using recombinant-p53 specific primers, wasthen used to detect p53 MRNA in the p53 treated tumors (FIG. 9, lanes1,2,4,5). No p53 signal was evident from the tumors excised from theβ-gal treated animals (FIG. 9, lanes 3 and 6). Amplification with actinprimers served as a control for the RT-PCR reaction (FIG. 9, lanes 7-9),while a plasmid containing the recombinant-p53 sequence served as apositive control for the recombinant-p53 specific band (FIG. 9, lane10). This experiment demonstrates that a p53 recombinant adenovirus canspecifically direct expression of p53 mRNA within established tumorsfollowing a single injection into the peritumoral space. It also showsin vivo viral persistence for at least one week following infection witha p53 recombinant adenovirus.

[0103] In Vivo Efficacy

[0104] To address the feasibility of gene therapy of established tumors,a tumor-bearing nude mouse model was used. H69 cells were injected intothe subcutaneous space on the right flank of mice, and tumors wereallowed to grow for 2 weeks. Mice then received peritumoral injectionsof buffer or recombinant virus twice weekly for a total of 8 doses. Inthe mice treated with buffer or control A/M virus, tumors continued togrow rapidly throughout the treatment, whereas those treated with theA/M/N/53 virus grew at a greatly reduced rate (FIG. 10A). Aftercessation of injections, the control treated tumors continued to growwhile the p53 treated tumors showed little or no growth for at least oneweek in the absence of any additional supply of exogenous p53 (FIG.10A). Although control animals treated with buffer alone had acceleratedtumor growth as compared to either virus treated group, no significantdifference in body weight was found between the three groups during thetreatment period. Tumor ulceration in some animals limited the relevanceof tumor size measurements after day 42. However, continued monitoringof the animals to determine survival time demonstrated a survivaladvantage for the p53-treated animals (FIG. 10B). The last of thecontrol adenovirus treated animals died on day 83, while buffer alonetreated controls had all expired by day 56. In contrast, all 5 animalstreated with the A/M/N/53 continue to survive (day 130 after cellinoculation) (FIG. 10B). Together, this data establish a p53-specificeffect on both tumor growth and survival time in animals withestablished p53-deficient tumors.

[0105] Adenovirus Vectors Expressing p53

[0106] Recombinant human adenovirus vectors which are capable ofexpressing high levels of wild-type p53 protein in a dose dependentmanner were constructed. Each vector contains deletions in the E1a andE1b regions which render the virus replication deficient (Challberg andKelly (1979); Horowitz, (1991)). Of further significance is that thesedeletions include those sequences encoding the E1b 19 and 55 kd protein.The 19 kd protein is reported to be involved in inhibiting apoptosis(White et al. (1992); Rao et al. (1992)), whereas the 55 kd protein isable to bind wild-type p53 protein (Sarnow et al. (1982); Heuvel et al.(1990)). By deleting these adenoviral sequences, potential inhibitors ofp53 function were removed through direct binding to p53 or potentialinhibition of p53 mediated apoptosis. Additional constructs were madewhich have had the remaining 3′ E1b sequence, including all protein IXcoding sequence, deleted as well. Although this has been reported toreduce the packaging size capacity of adenovirus to approximately 3 kbless than wild-type virus (Ghosh-Choudhury et al. (1987)), theseconstructs are also deleted in the E3 region so that the A/M/N/53 andA/C/N/53 constructs are well within this size range. By deleting the pIXregion, adenoviral sequences homologous to those contained in 293 cellsare reduced to approximately 300 base pairs, decreasing the chances ofregenerating replication-competent, wild-type adenovirus throughrecombination. Constructs lacking pIX coding sequence appear to haveequal efficacy to those with pIX.

[0107] p53/Adenovirus Efficacy In Vitro

[0108] In concordance with a strong dose dependency for expression ofp53 protein in infected cells, a dose-dependent, p53-specific inhibitionof tumor cell growth was demonstrated. Cell division, was inhibited anddemonstrated by the inhibition of DNA synthesis, in a wide variety oftumor cell types known to lack wild-type p53 protein expression.Bacchetti and Graham (1993) recently reported p53 specific inhibition ofDNA synthesis in the ovarian carcinoma cell line SKOV-3 by a p53recombinant adenovirus in similar experiments. In addition to ovariancarcinoma, additional human tumor cell lines were demonstrated,representative of clinically important human cancers and including linesover-expressing mutant p53 protein, can also be growth inhibited by thep53 recombinants of this invention. At MOIs where the A/C/N/53recombinant is 90-100% effective in inhibiting DNA synthesis in thesetumor types, control adenovirus mediated suppression is less than 20%.

[0109] Although Feinstein et al. (1992) reported that re-introduction ofwild-type p53 could induce differentiation and increase the proportionof cells in G₁ versus S+G₂ for leukemic K562 cells, no p53 specificeffect was found in this line. Horvath and Weber (1988) have reportedthat human peripheral blood lymphocytes are highly nonpermissive toadenovirus infection. In separate experiments, the recombinantsignificantly infected the non-responding K562 cells with recombinantA/C/β-gal adenovirus, while other cell lines, including the control HepG2 line and those showing a strong p53 effect, were readily infectable.Thus, at least part of the variability of efficacy would appear to bedue to variability of infection, although other factors may be involvedas well.

[0110] The results observed with the A/M/N/53 virus in FIG. 8demonstrates that complete suppression is possible in an in vivoenvironment. The resumption of tumor growth in 2 out of 4, p53 treatedanimals at the lower MOI most likely resulted from a small percentage ofcells not initially infected with the p53 recombinant at this dose. Thecomplete suppression seen with A/M/N/53 at the higher dose, however,shows that the ability of tumor growth to recover can be overcome.

[0111] p53/Adenovirus In Vivo Efficacy

[0112] Work presented here and by other groups (Chen et al. (1990);Takahashi et al. (1992)) have shown that human tumor cells lackingexpression of wild-type p53 can be treated ex vivo with p53 and resultin suppression of tumor growth when the treated cells are transferredinto an animal model. Applicants present the first evidence of tumorsuppressor gene therapy of an in vivo established tumor, resulting inboth suppression of tumor growth and increased survival time. InApplicants' system, delivery to tumor cells did not rely on directinjection into the tumor mass. Rather, p53 recombinant adenovirus wasinjected into the peritumoral space, and p53 mRNA expression wasdetected within the tumor. p53 expressed by the recombinants wasfunctional and strongly suppressed tumor growth as compared to that ofcontrol, non-p53 expressing adenovirus treated tumors. However, both p53and control virus treated tumor groups showed tumor suppression ascompared to buffer treated controls. It has been demonstrated that localexpression of tumor necrosis factor (TNF), interferon-γ), interleukin(IL)-2, IL-4 or IL-7 can lead to T-cell independent transient tumorsuppression in nude mice (Hoch et al. (1992)). Exposure of monocytes toadenovirus virions are also weak inducers of IFN-α/β (reviewed inGooding and Wold (1990)). Therefore, it is not surprising that sometumor suppression in nude mice was observed even with the controladenovirus. This virus mediated tumor suppression was not observed inthe ex vivo control virus treated Saos-2 tumor cells described earlier.The p53-specific in vivo tumor suppression was dramatically demonstratedby continued monitoring of the animals in FIG. 10. The survival time ofthe p53-treated mice was significantly increased, with 5 out of 5animals still alive more than 130 days after cell inoculation comparedto 0 out of 5 adenovirus control treated animals. The surviving animalsstill exhibit growing tumors which may reflect cells not initiallyinfected with the p53 recombinant adenovirus. Higher or more frequentdosing schedules may address this. In addition, promoter shutoff (Palmeret al. (1991)) or additional mutations may have rendered these cellsresistant to the p53 recombinant adenovirus treatment. For example,mutations in the recently described WAF1 gene, a gene induced bywild-type p53 which subsequently inhibits progression of the cell cycleinto S phase, (El-Deiry et al. (1993); Hunter (1993)) could result in ap53-resistant tumor.

EXPERIMENT NO. III

[0113] This Example shows the use of suicide genes and tissue specificexpression of such genes in the gene therapy methods described herein.Hepatocellular carcinoma was chosen as the target because it is one ofthe most common human malignancies affecting man, causing an estimated1,250,000 deaths per year world-wide. The incidence of this cancer isvery high in Southeast Asia and Africa where it is associated withHepatitis B and C infection and exposure to aflatoxin. Surgery iscurrently the only treatment which offers the potential for curing HCC,although less than 20% of patients are considered candidates forresection (Ravoet C. et al., 1993). However, tumors other thanhepatocellular carcinoma are equally applicable to the methods ofreducing their proliferation described herein.

[0114] Cell Lines

[0115] All cell lines but for the HLF cell line were obtained from theAmerican Type Tissue Culture Collection (ATCC) 12301 Parklawn Drive,Rockville Md. ATCC accession numbers are noted in parenthesis. The humanembryonal kidney cell line 293 (CRL 1573) was used to generate andpropagate the recombinant adenoviruses described herein. They weremaintained in DME medium containing 10% defined, supplemented calf serum(Hyclone). The hepatocellular carcinoma cell lines Hep 3B (HB 8064), HepG2 (HB 8065), and HLF were maintained in DME/F12 medium supplementedwith 10% fetal bovine serum, as were the breast carcinoma cell linesMDA-MB468 (HTB 132) and BT-549 (HTB 122). Chang liver cells (CCL 13)were grown in MEM medium supplemented with 10% fetal bovine serum. TheHLF cell line was obtained from Drs. T. Morsaki and H. Kitsuki at theKyushu University School of Medicine in Japan.

[0116] Recombinant Virus Construction

[0117] Two adenoviral expression vectors designated herein as ACNTK andAANTK and devoid of protein IX function (depicted in FIG. 11) arecapable of directing expression of the TK suicide gene within tumorcells. A third adenovirus expression vector designated AANCAT wasconstructed to further demonstrate the feasibility of specificallytargeting gene expression to specific cell types using adenoviralvectors. These adenoviral constructs were assembled as depicted in FIGS.11 and 12 and are derivatives of those previously described for theexpression of tumor suppresor genes.

[0118] For expression of the foreign gene, expression cassettes havebeen inserted that utilize either the human cytomegalovirus immediateearly promoter/enhancer (CMV) (Boshart, M. et al., 1985) or the humanalpha-fetoprotein (AFP) enhancer/promoter (Watanable, K. et al., 1987;Nakabayashi, H. et al., 1989) to direct transcription of the TK gene orthe chloramphenicol acetyltransferase gene (CAT). The CMV enhancerpromoter is capable of directing robust gene expression in a widevariety of cell types while the AFP enhancer/promoter constructrestricts expression to hepatocellular carcinoma cells (HCC) whichexpress AFP in about 70-80% of the HCC pateint population. In theconstruct utilizing the CMV promoter/enhancer, the adenovirus type 2tripartite leader sequence also was inserted to enhance translation ofthe TK transcript (Berkner, K. L. and Sharp, 1985). In addition to theE1 deletion, both adenovirus vectors are additionally deleted for 1.9kilobases (kb) of DNA in the viral E3 region. The DNA deleted in the E3region is non-essential for virus propagation and its deletion increasesthe insert capacity of the recombinant virus for foreign DNA by anequivalent amount (1.9 kb) (Graham and Prevec, 1991).

[0119] To demonstrate the specificity of the AFP promoter/enhancer, thevirus AANCAT also was constructed where the marker gene chloramphenicolaceytitransferase (CAT) is under the control of the AFPenhancer/promoter. In the ACNTK viral construct, the Ad2 tripartiteleader sequence was placed between the CMV promoter/enhancer and the TKgene. The tripartite leader has been reported to enhance translation oflinked genes. The E1 substitution impairs the ability of the recombinantviruses to replicate, restricting their propagation to 293 cells whichsupply the Ad5 E1 gene products in trans (Graham et al., 1977).

[0120] Adenoviral Vector ACNTK: The plasmid pMLBKTK in E. coli HB101(from ATCC #39369) was used as the source of the herpes simplex virus(HSV-1) thymidine kinase (TK) gene. TK was excised from this plasmid asa 1.7 kb gene fragment by digestion with the restriction enzymes Bgl IIand Pvu II and subcloned into the compatible Bam HI, EcoR V restrictionsites of plasmid pSP72 (Promega) using standard cloning techniques(Sambrook et al., 1989). The TK insert was then isolated as a 1.7 kbfragment from this vector by digestion with Xba I and Bgl II and clonedinto Xba I, BamHI digested plasmid pACN (Wills et al. 1994). Twenty (20)μg of this plasmid designated pACNTK were linearized with Eco RI andcotransfected into 293 cells (ATCC CRL 1573) with 5 μg of Cla I digestedACBGL (Wills et al., 1994 supra) using a CaPO₄ transfection kit(Stratagene, San Diego, Calif.). Viral plaques were isolated andrecombinants, designated ACNTK, were identified by restriction digestanalysis of isolated DNA with Xho I and BsiWI. Positive recombinantswere further purified by limiting dilution and expanded and titered bystandard methods (Graham and Prevec, 1991).

[0121] Adenoviral Vector AANTK: The α-fetoprotein promoter (AFP-P) andenhancer (AFP-E) were cloned from a human genomic DNA (Clontech) usingPCR amplification with primers containing restriction sites at theirends. The primers used to isolate the 210 bp AFP-E contained a Nhe Irestriction site on the 5′ primer and an Xba I, Xho I, Kpn I linker onthe 3′ primer. The 5′ primer sequence was 5′-CGC GCT AGC TCT GCC CCA AAGAGC T-3. The 5′ primer sequence was 5′-CGC GGT ACC CTC GAG TCT AGA TATTGC CAG TGG TGG AAG-3′. The primers used to isolate the 1763 bp AFEfragment contained a Not I restriction site on the 5′ primer and a Xba Isite on the 3′ primer. The 5′ primer sequence was 5′-CGT GCG GCC GCT GGAGGA CTT TGA GGA TGT CTG TC-3′. The 3′ primer sequence was 5′-CGC TCT AGAGAG ACC AGT TAG GAA GTT TTC GCA-3′. For PCR amplification, the DNA wasdenatured at 97° for 7 minutes, followed by 5 cycles of amplification at97°, 1 minute, 53°, 1 minute, 72°, 2 minutes, and a final 72°, 10 minuteextension. The amplified AFE was digested with Not I and Xba I andinserted into the Not I, Xba I sites of a plasmid vector (pA/ITR/B)containing adenovirus type 5 sequences 1-350 and 3330-5790 separated bya polylinker containing Not I, Xho I, Xba I, Hind III, Kpn I, Bam HI,Nco I, Sma I, and Bgl II sites. The amplified AFP-E was digested withNhe I and Kpn I and inserted into the AFP-E containing constructdescribed above which had been digested with Xba I and Kpn I. This newconstruct was then further digested with Xba I and NgoMI to removeadenoviral sequences 3330-5780, which were subsequently replaced with anXba I, NgoMI restriction fragment of plasmid pACN containing nucleotides4021-10457 of adenovirus type 2 to construct the plasmid pAAN containingboth the α-fetoprotein enhancer and promoter. This construct was thendigested with Eco RI and Xba I to isolate a 2.3 kb fragment containingthe Ad5 inverted terminal repeat, the AFP-E and the AFP-P which wassubsequently ligated with the 8.55 kb fragment of Eco RI, Xba I digestedpACNTK described above to generate pAANTK where the TK gene is driven bythe α-fetoprotein enhancer and promoter in an adenovirus background.This plasmid was then linearized with Eco RI and cotransfected with thelarge fragment of Cla I digested ALBGL as above and recombinants,designated AANTK, were isolated and purified as described above.

[0122] Adenoviral Vector AANCAT: The chloramphenicol acetyltransferase(CAT) gene was isolated from the pCAT-Basic Vector (Promega Corporation)by an Xba I, Bam HI digest. This 1.64 kb fragment was ligated into XbaI, Bam HI digested pAAN (described above) to create pAANCAT. Thisplasmid was then linearized with Eco RI and cotransfected with the largefragment of Cla I digested rA/C/β-gal to create AANCAT.

[0123] Reporter Gene Expression: βGalactosidase Expression:

[0124] Cells were plated at 1×10⁵ cells/well in a 24-well tissue cultureplate (Costar) and allowed to adhere overnight (37C., 7% CO₂). Overnightinfections of ACBGL were performed at a multiplicity of infection (MOI)of 30. After 24 hours, cells were fixed with 3.7% Formaldehyde; PBS, andstained with 1 mg/ml Xgal reagent (USB). The data was scored (+,++,+++)by estimating the percentage of positively stained cells at each MOI.[+=1-33%, ++=33-67% and +++=>67%]

[0125] Reported Gene Expression: CAT Expression:

[0126] Two (2)×10⁶ cells (Hep G2, Hep 3B, HLF, Chang, and MDA-MB468)were seeded onto 10 cm plates in triplicate and incubated overnight(37C., 7% CO₂). Each plate was then infected with either AANCAT at anMOI=30 or 100 or uninfected and allowed to incubate for 3 days. Thecells were then trypsinized and washed with PBS and resuspended in 100μl of 0.25 M Tris pH 7.8. The samples were frozen and thawed 3 times,and the supernatant was transferred to new tubes and incubated at 60° C.for 10 minutes. The samples were then spun at 4° C. for 5 minutes, andthe supernatants assayed for protein concentration using a Bradfordassay (Bio-Rad Protein Assay Kit). Samples were adjusted to equalprotein concentrations to a final volume of 75 μl using 0.25 M Tris, 25μl of 4 mM acetyl CoA and 1 μl of ¹⁴C-Chloramphenicol and incubatedovernight at 37° C. 500 μl of ethyl acetate is added to each sample andmixed by vortexing, followed by centrifiguration for 5 minutes at roomtemperature. The upper phase is then transferred to a new tube and theethyl acetate is evaporated by centrifugation under vacuum. The reactionproducts are then redissolved in 25 μl of ethyl acetate and spotted ontoa thin layer chromatography (TLC) plate and the plate is then placed ina pre-equilibrated TLC chamber (95% chloroform, 5% methanol). Thesolvent is then allowed to migrate to the top of the plate, the plate isthen dried and exposed to X-ray film.

[0127] Cellular Proliferation: ³H-Thymidine Incorporation

[0128] Cells were plated at 5×10³ cells/well in a 96-well micro-titerplate (Costar) and allowed to incubate overnight (37C., 7% CO₂) Seriallydiluted ACN, ACNTK or AATK virus in DMEM; 15% FBS; 1% glutamine was usedto transfect cells at an infection multiplicity of 30 for an overnightduration at which point cells were dosed in triplicate with ganciclovir(Cytovene) at log intervals between 0.001 and 100 mM (micro molar). 1μCi ³H-thymidine (Amersham) was added to each well 12-18 hours beforeharvesting. At 72 hours-post infection cells were harvested ontoglass-fiber filters and incorporated ³H-thymidine was counted usingliquid scintillation (TopCount, Packard). Results are plotted as percentof untreated control proliferation and tabulated as the effective dose(ED₅₀±SD) for a 50 percent reduction in proliferation over mediacontrols. ED₅₀ values were estimated by fitting a logistic equation tothe dose response data.

[0129] Cytotoxicity: LDH Release

[0130] Cells (HLF, human HCC) were plated, infected with ACN or ACNTKand treated with ganciclovir as described for the proliferation assay.At 72 hours post-ganciclovir administration, cells were spun, thesupernatant was removed. The levels of lactate dehydrogenase measuredcolometrically (Promega, Cytotox 96™). Mean (+/−S.D.) LDH release isplotted versus M.O.I.

[0131] IN VIVO THERAPY

[0132] Human hepatocellular carcinoma cells (Hep 3B) were injectedsubcutaneously into ten female (10) athymic nu/nu mice (SimonsenLaboratories, Gilroy, Calif.). Each animal received approximately 1×10⁷cells in the left flank. Tumors were allowed to grow for 27 days beforerandomizing mice by tumor size. Mice were treated with intratumoral andperitumoral injections of ACNTK or the control virus ACN (1×10⁹ iu in100 μl) every other day for a total of three doses. Starting 24 hoursafter the initial dose of adenovirus, the mice were dosedintraperitoneally with ganciclovir (Cytovene 100 mg/kg) daily for atotal of 10 days. Mice were monitored for tumor size and body weighttwice weekly. Measurements on tumors were made in three dimensions usingvernier calipers and volumes were calculated using the formula 4/3πr³,where r is one-half the average tumor dimension.

[0133] Results

[0134] The recombinant adenoviruses were used to infect three HCC celllines (HLF, Hep3B and Hep-G2). One human liver cell line (Chang) and twobreast cancer cell lines were used as controls (MDAMB468 and BT549). Todemonstrate the specificity of the AFP promoter/enhancer, the virusAANCAT was constructed. This virus was used to infect cells that eitherdo (Hep 3B, HepG2) or do not (HLE, Chang, MDAMB468) express the HCCtumor marker alpha-fetoprotein (AFP). As shown in FIG. 13, AANCATdirects expression of the CAT marker gene only in those HCC cells whichare capable of expressing AFP (FIG. 13).

[0135] The efficacy of ACNTK and AANTK for the treatment of HCC wasassessed using a ³H-thymidine incorporation assay to measure the effectof the combination of HSV-TK expression and ganciclovir treatment uponcellular proliferation. The cell lines were infected with either ACNTKor AANTK or the control virus ACN (Wills et al., 1994 supra), which doesnot direct expression of HSV-TK, and then treated with increasingconcentrations of ganciclovir. The effect of this treatment was assessedas a function of increasing concentrations of ganciclovir, and theconcentration of ganciclovir required to inhibit ³H-thymidineincorporated by 50% was determined (ED₅₀). Additionally, a relativemeasure of adenovirus—mediated gene transfer and expression of each cellline was determined using a control virus which directs expression ofthe marker gene beta-galactosidase. The data presented in FIG. 14 andTable 1 below show that the ACNTK virus/ganciclovir combinationtreatment was capable of inhibiting cellular proliferation in all celllines examined as compared with the control adenovirus ACN incombination with ganciclovir. In contrast, the AANTK viral vector wasonly effective in those HCC cell lines which have been demonstrated toexpress α-fetoprotein. In addition, the AANTK/GCV combination was moreeffective when the cells were plated at high densities. TABLE 1 β-galED50 Cell Line aFP Expression ACN ACNTK AANTK MDAMB468 − +++ >100 2 >100BT549 − +++ >100 <0.3 >100 HLF − +++ >100 0.8 >100 CHANG − +++ >10022 >100 HEP-3B − + 80 8 8 HEP-G2 LOW + ++ 90 2 35  HEP-G2 HIGH + ++ 890.5 4

[0136] Nude mice bearing Hep3B tumors (N=5/group) were treatedintratumorally and peritumorally with equivalent doses of ACNTK or ACNcontrol. Twenty-four hours after the first administration of recombinantadenovirus, daily treatment of ganciclovir was initiated in all mice.Tumor dimensions from each animal were measured twice weekly viacalipers, and average tumor sizes are plotted in FIG. 16. Average tumorsize at day 58 was smaller in the ACNTK-treated animals but thedifference did not reach statistical significance (p<0.09, unpairedt-test). These data support a specific effect of ACNTK on tumor growthin vivo. No significant differences in average body weight were detectedbetween the groups.

[0137] Although the invention has been described with reference to theabove embodiments, it should be understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the claims that follow.

[0138] References

[0139] AIELLO, L. et al. (1979) Virology 94:460-469.

[0140] AMERICAN CANCER SOCIETY. (1993) Cancer Facts and Figures.

[0141] AULITZKY et al. (1991) Eur. J. Cancer 27(4) :462-467.

[0142] AUSTIN, E. A. and HUBER, B. E. (1993) Mol. Pharmaceutical43:380-387.

[0143] BACCHETTI, S. AND GRAHAM, F. (1993) International Journal ofOncology 3:781-788.

[0144] BAKER S. J., MARKOWITZ, S., FEARON E. R., WILLSON, J. K. V., ANDVOGELSTEIN, B. (1990) Science 249:912-915.

[0145] BARTEK, J., BARTKOVA, J., VOJTESEK, B., STASKOVA, Z., LUKAS, J.,REJTHAR, A., KOVARIK, J., MIDGLEY, C. A., GANNON, J. V., AND LANE, D. P.(1991) Oncogene 6:1699-1703.

[0146] BERKNER, K. L. and SHARP (1985) Nucleic Acids Res 13:841-857.

[0147] BOSHART, M. et al. (1985) Cell 41:521-530.

[0148] BRESSAC, B., GALVIN, K. M., LIANG, T. J., ISSELBACHER, K. J.,WANDS, J. R., AND OZTURK, M. (1990) Proc. Natl. Acad. Sci. USA87:1973-1977.

[0149] CARUSO M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:7024-7028.

[0150] CHALLBERG, M. D., KELLY, T. J. (1979) Biochemistry 76:655-659.

[0151] CHEN P. L., CHEN Y., BOOKSTEIN R., AND LEE W. H. (1990) Science250:1576-1580.

[0152] CHEN, Y., CHEN, P. L., ARNAIZ, N., GOODRICH, D., AND LEE, W. H.(1991) Oncogene 6:1799-1805.

[0153] CHENG, J L, YEE, J. K., YEARGIN, J., FRIEDMANN, T., AND HAAS, M.(1992) Cancer Research 52:222-226.

[0154] COLBY, W. W. AND SHENK, T. J. (1981) Virology 39:977-980.

[0155] CULVER ET AL. (1991) P.N.A.S. (U.S.A.) 88:3155-3159.

[0156] CULVER, K. W. et al. (1992) Science 256:1550-1552.

[0157] DEMETRI et al. (1989) J. Clin. Oncol. 7(10):1545-1553.

[0158] DILLER, L., et al. (1990) Mol. Cell. Biology 10:5772-5781.

[0159] EL-DEIRY, W. S., et al. (1993) Cell 75:817-825.

[0160] EZZIDINE, Z. D. et al. (1991) The New Biologist 3:608-614.

[0161] FEINSTEIN, E., GALE, R. P., REED, J., AND CANAANI, E. (1992)Oncogene 7:1853-1857.

[0162] GHOSH-CHOUDHURY, G., HAJ-AHMAD, Y., AND GRAHAM, F. L. (1987) EMBOJournal 6:1733-1739.

[0163] GOODING, L. R., AND WOLD, W. S. M. (1990) Crit. Rev. Immunol.10:53-71.

[0164] GRAHAM F. L., AND VAN DER ERB A. J. (1973) Virology 52:456-467.

[0165] GRAHAM, F. L. AND PREVEC, L. (1992) Vaccines: New Approaches toImmunological Problems. R. W. Ellis (ed), Butterworth-Heinemann, Boston.pp. 363-390.

[0166] GRAHAM, F. L., SMILEY, J., RUSSELL, W. C. AND NAIRN, R. (1977) J.Gen. Virol. 36:59-74.

[0167] GRAHAM F. L. AND PREVEC L. (1991) Manipulation of adenovirusvectors. In: Methods in Molecular Biology. Vol 7: Gene Transfer andExpression Protocols. Murray E. J. (ed.) The Humana Press Inc., CliftonN. J., Vol 7:109-128.

[0168] HEUVEL, S. J. L., LAAR, T., KAST, W. M., MELIEF, C. J. M.,ZANTEMA, A., AND VAN DER EB, A. J. (1990) EMBO Journal 9:2621-2629.

[0169] HOCK, H., DORSCH, M., KUZENDORF, U., QIN, Z., DIAMANTSTEIN, T.,AND BLANKENSTEIN, T. (1992) Proc. Natl. Acad. Sci. USA 90:2774-2778.

[0170] HOLLSTEIN, M., SIDRANSKY, D., VOGELSTEIN, B., AND HARRIS, C.(1991) Science 253:49-53.

[0171] HOROWITZ, M. S. (1991) Adenoviridae and their replication. InFields Virology. B. N. Fields, ed. (Raven Press, New York) pp.1679-1721.

[0172] HORVATH, J., AND WEBER, J. M. (1988) J. Virol. 62:341-345.

[0173] HUANG et al. (1991) Nature 350:160-162.

[0174] HUBER, B. E. et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043.

[0175] HUNTER, T. (1993) Cell 75:839-841.

[0176] JONES, N. AND SHENK, T. (1979) Cell 17:683-689.

[0177] KAMB et al. (1994) Science 264:436-440.

[0178] KEURBITZ, S. J., PLUNKETT, B. S., WALSH, W. V., AND KASTAN, M. B.(1992) Proc. Natl. Acad. Sci. USA 89: 7491-7495.

[0179] KREIGLER, M. Gene Transfer and Expression: A Laboratory Manual,W. H. Freeman and Company, New York (1990).

[0180] LANDMANN et al. (1992) J. Interferon Res. 12(2):103-111.

[0181] LANE, D. P. (1992) Nature 358:15-16.

[0182] LANTZ et al. (1990) Cytokine 2(6) :402-406.

[0183] LARRICK, J. W. and BURCK, K. L. Gene Therapy: Application ofMolecular Biology, Elsevier Science Publishing Co., Inc. New York, N.Y.(1991).

[0184] LEE et al. (1987) Science 235:1394-1399.

[0185] LEMAISTRE et al. (1991) Lancet 337:1124-1125.

[0186] LEMARCHAND, P., et al. (1992) Proc. Natl. Acad. Sci. USA89:6482-6486.

[0187] LEVINE, A. J. (1993) The Tumor Suppressor Genes. Annu. Rev.Biochem. 1993. 62:623-651.

[0188] LOWE S. W., SCHMITT, E. M., SMITH, S. W., OSBORNE, B. A., ANDJACKS, J. (1993) Nature 362:847-852.

[0189] LOWE, S. W., RULEY, H. E., JACKS, T., AND HOUSMAN, D. E. (1993)Cell 74:957-967.

[0190] MARTIN (1975) In: Remington's Pharm. Sci., 15th Ed. (Mack Publ.Co., Easton).

[0191] MERCER, W. E., et al. (1990) Proc. Natl. Acad. Sci. USA87:6166-6170.

[0192] NAKABAYASHI, H. et al. (1989) The Journal of Biological Chemistry264:266-271.

[0193] PALMER, T. D., ROSMAN, G. J., OSBORNE, W. R., AND MILLER, A. D.(1991) Proc. Natl. Acad. Sci USA 88:1330-1334.

[0194] RAO, L., DEBBAS, M., SABBATINI, P., HOCKENBERY, D., KORSMEYER,S., AND WHITE, E. (1992) Proc. Natl. Acad. Sci. USA 89:7742-7746.

[0195] RAVOET C. et al. (1993) Journal of Surgical Oncology Supplement3:104-111.

[0196] RICH, D. P., et al. (1993) Human Gene Therapy 4:460-476.

[0197] ROSENFELD, M. A., et al. (1992) Cell 68:143-155.

[0198] SAMBROOK J., FRITSCH E. F., AND MANIATIS T. (1989). MolecularCloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor).

[0199] SARNOW, P., HO, Y. S., WILLIAMS, J., AND LEVINE, A. J. (1982)Cell 28:387-394.

[0200] SHAW, P., BOVEY, R., TARDY, S., SAHLI, R., SORDAT, B., AND COSTA,J. (1992) Proc. Natl. Acad. Sci. USA 89:4495-4499.

[0201] SIEGFRIED, W. (1993) Exp. Clin. Endocrinol. 101:7-11.

[0202] SORSCHER, E. J. et al. (1994) Gene Therapy 1:233-238.

[0203] SPECTOR, D. J. (1983) Virology 130:533-538.

[0204] STEWART, P. L. et al. (1993) EMBO Journal 12:2589-2599.

[0205] STRAUS. S. E. (1984) Adenovirus infections in humans. In: TheAdenoviruses, Ginsberg HS, ed. New York: Plenum Press, 451-496.

[0206] SUPERSAXO et al. (1988) Pharm. Res. 5(8):472-476.

[0207] TAKAHASHI, T., et al. (1989) Science 246: 491-494.

[0208] TAKAHASHI, T., et al. (1992) Cancer Research 52:2340-2343.

[0209] THIMMAPPAYA, B. et al. (1982) Cell 31:543-551.

[0210] WANG, A. M., DOYLE, M. V., AND MARK, D. F. (1989) Proc. Natl.Acad. Sci USA 86:9717-9721.

[0211] WATANABLE, K. et al. (1987) The Journal of Biological Chemistry262:4812-4818.

[0212] WHITE, E., et al. (1992) Mol. Cell. Biol. 12:2570-2580.

[0213] WILLS, K. N. et al. (1994) Hum. Gen. Ther. 5:1079-1088.

[0214] YONISH-ROUACH, E., et al. (1991) Nature 352:345-347.

1 9 25 base pairs nucleic acid single linear DNA 1 CGCCACCGAG GGACCTGAGCGAGTC 25 20 base pairs nucleic acid single linear DNA 2 TTCTGGGAAGGGACAGAAGA 20 25 base pairs nucleic acid single linear DNA 3 CGCGCTAGCTCTGCCCCAAA GAGCT 25 39 base pairs nucleic acid single linear DNA 4CGCGGTACCC TCGAGTCTAG ATATTGCCAG TGGTGGAAG 39 35 base pairs nucleic acidsingle linear DNA 5 CGTGCGGCCG CTGGAGGACT TTGAGGATGT CTGTC 35 33 basepairs nucleic acid single linear DNA 6 CGCTCTAGAG AGACCAGTTA GGAAGTTTTCGCA 33 2995 base pairs nucleic acid single linear cDNA CDS 139..2925/product= “RB” /note= “retinoblastoma tumor suppressor” 7 TTCCGGTTTTTCTCAGGGGA CGTTGAAATT ATTTTTGTAA CGGGAGTCGG GAGAGGACGG 60 GGCGTGCCCCGCGTGCGCGC GCGTCGTCCT CCCCGGCGCT CCTCCACAGC TCGCTGGCTC 120 CCGCCGCGGAAAGGCGTC ATG CCG CCC AAA ACC CCC CGA AAA ACG GCC GCC 171 Met Pro Pro LysThr Pro Arg Lys Thr Ala Ala 1 5 10 ACC GCC GCC GCT GCC GCC GCG GAA CCCCCG GCA CCG CCG CCG CCG CCC 219 Thr Ala Ala Ala Ala Ala Ala Glu Pro ProAla Pro Pro Pro Pro Pro 15 20 25 CCT CCT GAG GAG GAC CCA GAG CAG GAC AGCGGC CCG GAG GAC CTG CCT 267 Pro Pro Glu Glu Asp Pro Glu Gln Asp Ser GlyPro Glu Asp Leu Pro 30 35 40 CTC GTC AGG CTT GAG TTT GAA GAA ACA GAA GAACCT GAT TTT ACT GCA 315 Leu Val Arg Leu Glu Phe Glu Glu Thr Glu Glu ProAsp Phe Thr Ala 45 50 55 TTA TGT CAG AAA TTA AAG ATA CCA GAT CAT GTC AGAGAG AGA GCT TGG 363 Leu Cys Gln Lys Leu Lys Ile Pro Asp His Val Arg GluArg Ala Trp 60 65 70 75 TTA ACT TGG GAG AAA GTT TCA TCT GTG GAT GGA GTATTG GGA GGT TAT 411 Leu Thr Trp Glu Lys Val Ser Ser Val Asp Gly Val LeuGly Gly Tyr 80 85 90 ATT CAA AAG AAA AAG GAA CTG TGG GGA ATC TGT ATC TTTATT GCA GCA 459 Ile Gln Lys Lys Lys Glu Leu Trp Gly Ile Cys Ile Phe IleAla Ala 95 100 105 GTT GAC CTA GAT GAG ATG TCG TTC ACT TTT ACT GAG CTACAG AAA AAC 507 Val Asp Leu Asp Glu Met Ser Phe Thr Phe Thr Glu Leu GlnLys Asn 110 115 120 ATA GAA ATC AGT GTC CAT AAA TTC TTT AAC TTA CTA AAAGAA ATT GAT 555 Ile Glu Ile Ser Val His Lys Phe Phe Asn Leu Leu Lys GluIle Asp 125 130 135 ACC AGT ACC AAA GTT GAT AAT GCT ATG TCA AGA CTG TTGAAG AAG TAT 603 Thr Ser Thr Lys Val Asp Asn Ala Met Ser Arg Leu Leu LysLys Tyr 140 145 150 155 GAT GTA TTG TTT GCA CTC TTC AGC AAA TTG GAA AGGACA TGT GAA CTT 651 Asp Val Leu Phe Ala Leu Phe Ser Lys Leu Glu Arg ThrCys Glu Leu 160 165 170 ATA TAT TTG ACA CAA CCC AGC AGT TCG ATA TCT ACTGAA ATA AAT TCT 699 Ile Tyr Leu Thr Gln Pro Ser Ser Ser Ile Ser Thr GluIle Asn Ser 175 180 185 GCA TTG GTG CTA AAA GTT TCT TGG ATC ACA TTT TTATTA GCT AAA GGG 747 Ala Leu Val Leu Lys Val Ser Trp Ile Thr Phe Leu LeuAla Lys Gly 190 195 200 GAA GTA TTA CAA ATG GAA GAT GAT CTG GTG ATT TCATTT CAG TTA ATG 795 Glu Val Leu Gln Met Glu Asp Asp Leu Val Ile Ser PheGln Leu Met 205 210 215 CTA TGT GTC CTT GAC TAT TTT ATT AAA CTC TCA CCTCCC ATG TTG CTC 843 Leu Cys Val Leu Asp Tyr Phe Ile Lys Leu Ser Pro ProMet Leu Leu 220 225 230 235 AAA GAA CCA TAT AAA ACA GCT GTT ATA CCC ATTAAT GGT TCA CCT CGA 891 Lys Glu Pro Tyr Lys Thr Ala Val Ile Pro Ile AsnGly Ser Pro Arg 240 245 250 ACA CCC AGG CGA GGT CAG AAC AGG AGT GCA CGGATA GCA AAA CAA CTA 939 Thr Pro Arg Arg Gly Gln Asn Arg Ser Ala Arg IleAla Lys Gln Leu 255 260 265 GAA AAT GAT ACA AGA ATT ATT GAA GTT CTC TGTAAA GAA CAT GAA TGT 987 Glu Asn Asp Thr Arg Ile Ile Glu Val Leu Cys LysGlu His Glu Cys 270 275 280 AAT ATA GAT GAG GTG AAA AAT GTT TAT TTC AAAAAT TTT ATA CCT TTT 1035 Asn Ile Asp Glu Val Lys Asn Val Tyr Phe Lys AsnPhe Ile Pro Phe 285 290 295 ATG AAT TCT CTT GGA CTT GTA ACA TCT AAT GGACTT CCA GAG GTT GAA 1083 Met Asn Ser Leu Gly Leu Val Thr Ser Asn Gly LeuPro Glu Val Glu 300 305 310 315 AAT CTT TCT AAA CGA TAC GAA GAA ATT TATCTT AAA AAT AAA GAT CTA 1131 Asn Leu Ser Lys Arg Tyr Glu Glu Ile Tyr LeuLys Asn Lys Asp Leu 320 325 330 GAT GCA AGA TTA TTT TTG GAT CAT GAT AAAACT CTT CAG ACT GAT TCT 1179 Asp Ala Arg Leu Phe Leu Asp His Asp Lys ThrLeu Gln Thr Asp Ser 335 340 345 ATA GAC AGT TTT GAA ACA CAG AGA ACA CCACGA AAA AGT AAC CTT GAT 1227 Ile Asp Ser Phe Glu Thr Gln Arg Thr Pro ArgLys Ser Asn Leu Asp 350 355 360 GAA GAG GTG AAT GTA ATT CTT CCA CAC ACTCCA GTT AGG ACT GTT ATG 1275 Glu Glu Val Asn Val Ile Leu Pro His Thr ProVal Arg Thr Val Met 365 370 375 AAC ACT ATC CAA CAA TTA ATG ATG ATT TTAAAT TCA GCA AGT GAT CAA 1323 Asn Thr Ile Gln Gln Leu Met Met Ile Leu AsnSer Ala Ser Asp Gln 380 385 390 395 CCT TCA GAA AAT CTG ATT TCC TAT TTTAAC AAC TGC ACA GTG AAT CCA 1371 Pro Ser Glu Asn Leu Ile Ser Tyr Phe AsnAsn Cys Thr Val Asn Pro 400 405 410 AAA GAA AGT ATA CTG AAA AGA GTG AAGGAT ATA GGA TAC ATC TTT AAA 1419 Lys Glu Ser Ile Leu Lys Arg Val Lys AspIle Gly Tyr Ile Phe Lys 415 420 425 GAG AAA TTT GCT AAA GCT GTG GGA CAGGGT TGT GTC GAA ATT GGA TCA 1467 Glu Lys Phe Ala Lys Ala Val Gly Gln GlyCys Val Glu Ile Gly Ser 430 435 440 CAG CGA TAC AAA CTT GGA GTT CGC TTGTAT TAC CGA GTA ATG GAA TCC 1515 Gln Arg Tyr Lys Leu Gly Val Arg Leu TyrTyr Arg Val Met Glu Ser 445 450 455 ATG CTT AAA TCA GAA GAA GAA CGA TTATCC ATT CAA AAT TTT AGC AAA 1563 Met Leu Lys Ser Glu Glu Glu Arg Leu SerIle Gln Asn Phe Ser Lys 460 465 470 475 CTT CTG AAT GAC AAC ATT TTT CATATG TCT TTA TTG GCG TGC GCT CTT 1611 Leu Leu Asn Asp Asn Ile Phe His MetSer Leu Leu Ala Cys Ala Leu 480 485 490 GAG GTT GTA ATG GCC ACA TAT AGCAGA AGT ACA TCT CAG AAT CTT GAT 1659 Glu Val Val Met Ala Thr Tyr Ser ArgSer Thr Ser Gln Asn Leu Asp 495 500 505 TCT GGA ACA GAT TTG TCT TTC CCATGG ATT CTG AAT GTG CTT AAT TTA 1707 Ser Gly Thr Asp Leu Ser Phe Pro TrpIle Leu Asn Val Leu Asn Leu 510 515 520 AAA GCC TTT GAT TTT TAC AAA GTGATC GAA AGT TTT ATC AAA GCA GAA 1755 Lys Ala Phe Asp Phe Tyr Lys Val IleGlu Ser Phe Ile Lys Ala Glu 525 530 535 GGC AAC TTG ACA AGA GAA ATG ATAAAA CAT TTA GAA CGA TGT GAA CAT 1803 Gly Asn Leu Thr Arg Glu Met Ile LysHis Leu Glu Arg Cys Glu His 540 545 550 555 CGA ATC ATG GAA TCC CTT GCATGG CTC TCA GAT TCA CCT TTA TTT GAT 1851 Arg Ile Met Glu Ser Leu Ala TrpLeu Ser Asp Ser Pro Leu Phe Asp 560 565 570 CTT ATT AAA CAA TCA AAG GACCGA GAA GGA CCA ACT GAT CAC CTT GAA 1899 Leu Ile Lys Gln Ser Lys Asp ArgGlu Gly Pro Thr Asp His Leu Glu 575 580 585 TCT GCT TGT CCT CTT AAT CTTCCT CTC CAG AAT AAT CAC ACT GCA GCA 1947 Ser Ala Cys Pro Leu Asn Leu ProLeu Gln Asn Asn His Thr Ala Ala 590 595 600 GAT ATG TAT CTT TCT CCT GTAAGA TCT CCA AAG AAA AAA GGT TCA ACT 1995 Asp Met Tyr Leu Ser Pro Val ArgSer Pro Lys Lys Lys Gly Ser Thr 605 610 615 ACG CGT GTA AAT TCT ACT GCAAAT GCA GAG ACA CAA GCA ACC TCA GCC 2043 Thr Arg Val Asn Ser Thr Ala AsnAla Glu Thr Gln Ala Thr Ser Ala 620 625 630 635 TTC CAG ACC CAG AAG CCATTG AAA TCT ACC TCT CTT TCA CTG TTT TAT 2091 Phe Gln Thr Gln Lys Pro LeuLys Ser Thr Ser Leu Ser Leu Phe Tyr 640 645 650 AAA AAA GTG TAT CGG CTAGCC TAT CTC CGG CTA AAT ACA CTT TGT GAA 2139 Lys Lys Val Tyr Arg Leu AlaTyr Leu Arg Leu Asn Thr Leu Cys Glu 655 660 665 CGC CTT CTG TCT GAG CACCCA GAA TTA GAA CAT ATC ATC TGG ACC CTT 2187 Arg Leu Leu Ser Glu His ProGlu Leu Glu His Ile Ile Trp Thr Leu 670 675 680 TTC CAG CAC ACC CTG CAGAAT GAG TAT GAA CTC ATG AGA GAC AGG CAT 2235 Phe Gln His Thr Leu Gln AsnGlu Tyr Glu Leu Met Arg Asp Arg His 685 690 695 TTG GAC CAA ATT ATG ATGTGT TCC ATG TAT GGC ATA TGC AAA GTG AAG 2283 Leu Asp Gln Ile Met Met CysSer Met Tyr Gly Ile Cys Lys Val Lys 700 705 710 715 AAT ATA GAC CTT AAATTC AAA ATC ATT GTA ACA GCA TAC AAG GAT CTT 2331 Asn Ile Asp Leu Lys PheLys Ile Ile Val Thr Ala Tyr Lys Asp Leu 720 725 730 CCT CAT GCT GTT CAGGAG ACA TTC AAA CGT GTT TTG ATC AAA GAA GAG 2379 Pro His Ala Val Gln GluThr Phe Lys Arg Val Leu Ile Lys Glu Glu 735 740 745 GAG TAT GAT TCT ATTATA GTA TTC TAT AAC TCG GTC TTC ATG CAG AGA 2427 Glu Tyr Asp Ser Ile IleVal Phe Tyr Asn Ser Val Phe Met Gln Arg 750 755 760 CTG AAA ACA AAT ATTTTG CAG TAT GCT TCC ACC AGG CCC CCT ACC TTG 2475 Leu Lys Thr Asn Ile LeuGln Tyr Ala Ser Thr Arg Pro Pro Thr Leu 765 770 775 TCA CCA ATA CCT CACATT CCT CGA AGC CCT TAC AAG TTT CCT AGT TCA 2523 Ser Pro Ile Pro His IlePro Arg Ser Pro Tyr Lys Phe Pro Ser Ser 780 785 790 795 CCC TTA CGG ATTCCT GGA GGG AAC ATC TAT ATT TCA CCC CTG AAG AGT 2571 Pro Leu Arg Ile ProGly Gly Asn Ile Tyr Ile Ser Pro Leu Lys Ser 800 805 810 CCA TAT AAA ATTTCA GAA GGT CTG CCA ACA CCA ACA AAA ATG ACT CCA 2619 Pro Tyr Lys Ile SerGlu Gly Leu Pro Thr Pro Thr Lys Met Thr Pro 815 820 825 AGA TCA AGA ATCTTA GTA TCA ATT GGT GAA TCA TTC GGG ACT TCT GAG 2667 Arg Ser Arg Ile LeuVal Ser Ile Gly Glu Ser Phe Gly Thr Ser Glu 830 835 840 AAG TTC CAG AAAATA AAT CAG ATG GTA TGT AAC AGC GAC CGT GTG CTC 2715 Lys Phe Gln Lys IleAsn Gln Met Val Cys Asn Ser Asp Arg Val Leu 845 850 855 AAA AGA AGT GCTGAA GGA AGC AAC CCT CCT AAA CCA CTG AAA AAA CTA 2763 Lys Arg Ser Ala GluGly Ser Asn Pro Pro Lys Pro Leu Lys Lys Leu 860 865 870 875 CGC TTT GATATT GAA GGA TCA GAT GAA GCA GAT GGA AGT AAA CAT CTC 2811 Arg Phe Asp IleGlu Gly Ser Asp Glu Ala Asp Gly Ser Lys His Leu 880 885 890 CCA GGA GAGTCC AAA TTT CAG CAG AAA CTG GCA GAA ATG ACT TCT ACT 2859 Pro Gly Glu SerLys Phe Gln Gln Lys Leu Ala Glu Met Thr Ser Thr 895 900 905 CGA ACA CGAATG CAA AAG CAG AAA ATG AAT GAT AGC ATG GAT ACC TCA 2907 Arg Thr Arg MetGln Lys Gln Lys Met Asn Asp Ser Met Asp Thr Ser 910 915 920 AAC AAG GAAGAG AAA TGAGGATCTC AGGACCTTGG TGGACACTGT GTACACCTCT 2962 Asn Lys Glu GluLys 925 GGATTCATTG TCTCTCACAG ATGTGACTGA TAT 2995 928 amino acids aminoacid linear protein 8 Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala ThrAla Ala Ala Ala 1 5 10 15 Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro ProPro Pro Glu Glu Asp 20 25 30 Pro Glu Gln Asp Ser Gly Pro Glu Asp Leu ProLeu Val Arg Leu Glu 35 40 45 Phe Glu Glu Thr Glu Glu Pro Asp Phe Thr AlaLeu Cys Gln Lys Leu 50 55 60 Lys Ile Pro Asp His Val Arg Glu Arg Ala TrpLeu Thr Trp Glu Lys 65 70 75 80 Val Ser Ser Val Asp Gly Val Leu Gly GlyTyr Ile Gln Lys Lys Lys 85 90 95 Glu Leu Trp Gly Ile Cys Ile Phe Ile AlaAla Val Asp Leu Asp Glu 100 105 110 Met Ser Phe Thr Phe Thr Glu Leu GlnLys Asn Ile Glu Ile Ser Val 115 120 125 His Lys Phe Phe Asn Leu Leu LysGlu Ile Asp Thr Ser Thr Lys Val 130 135 140 Asp Asn Ala Met Ser Arg LeuLeu Lys Lys Tyr Asp Val Leu Phe Ala 145 150 155 160 Leu Phe Ser Lys LeuGlu Arg Thr Cys Glu Leu Ile Tyr Leu Thr Gln 165 170 175 Pro Ser Ser SerIle Ser Thr Glu Ile Asn Ser Ala Leu Val Leu Lys 180 185 190 Val Ser TrpIle Thr Phe Leu Leu Ala Lys Gly Glu Val Leu Gln Met 195 200 205 Glu AspAsp Leu Val Ile Ser Phe Gln Leu Met Leu Cys Val Leu Asp 210 215 220 TyrPhe Ile Lys Leu Ser Pro Pro Met Leu Leu Lys Glu Pro Tyr Lys 225 230 235240 Thr Ala Val Ile Pro Ile Asn Gly Ser Pro Arg Thr Pro Arg Arg Gly 245250 255 Gln Asn Arg Ser Ala Arg Ile Ala Lys Gln Leu Glu Asn Asp Thr Arg260 265 270 Ile Ile Glu Val Leu Cys Lys Glu His Glu Cys Asn Ile Asp GluVal 275 280 285 Lys Asn Val Tyr Phe Lys Asn Phe Ile Pro Phe Met Asn SerLeu Gly 290 295 300 Leu Val Thr Ser Asn Gly Leu Pro Glu Val Glu Asn LeuSer Lys Arg 305 310 315 320 Tyr Glu Glu Ile Tyr Leu Lys Asn Lys Asp LeuAsp Ala Arg Leu Phe 325 330 335 Leu Asp His Asp Lys Thr Leu Gln Thr AspSer Ile Asp Ser Phe Glu 340 345 350 Thr Gln Arg Thr Pro Arg Lys Ser AsnLeu Asp Glu Glu Val Asn Val 355 360 365 Ile Leu Pro His Thr Pro Val ArgThr Val Met Asn Thr Ile Gln Gln 370 375 380 Leu Met Met Ile Leu Asn SerAla Ser Asp Gln Pro Ser Glu Asn Leu 385 390 395 400 Ile Ser Tyr Phe AsnAsn Cys Thr Val Asn Pro Lys Glu Ser Ile Leu 405 410 415 Lys Arg Val LysAsp Ile Gly Tyr Ile Phe Lys Glu Lys Phe Ala Lys 420 425 430 Ala Val GlyGln Gly Cys Val Glu Ile Gly Ser Gln Arg Tyr Lys Leu 435 440 445 Gly ValArg Leu Tyr Tyr Arg Val Met Glu Ser Met Leu Lys Ser Glu 450 455 460 GluGlu Arg Leu Ser Ile Gln Asn Phe Ser Lys Leu Leu Asn Asp Asn 465 470 475480 Ile Phe His Met Ser Leu Leu Ala Cys Ala Leu Glu Val Val Met Ala 485490 495 Thr Tyr Ser Arg Ser Thr Ser Gln Asn Leu Asp Ser Gly Thr Asp Leu500 505 510 Ser Phe Pro Trp Ile Leu Asn Val Leu Asn Leu Lys Ala Phe AspPhe 515 520 525 Tyr Lys Val Ile Glu Ser Phe Ile Lys Ala Glu Gly Asn LeuThr Arg 530 535 540 Glu Met Ile Lys His Leu Glu Arg Cys Glu His Arg IleMet Glu Ser 545 550 555 560 Leu Ala Trp Leu Ser Asp Ser Pro Leu Phe AspLeu Ile Lys Gln Ser 565 570 575 Lys Asp Arg Glu Gly Pro Thr Asp His LeuGlu Ser Ala Cys Pro Leu 580 585 590 Asn Leu Pro Leu Gln Asn Asn His ThrAla Ala Asp Met Tyr Leu Ser 595 600 605 Pro Val Arg Ser Pro Lys Lys LysGly Ser Thr Thr Arg Val Asn Ser 610 615 620 Thr Ala Asn Ala Glu Thr GlnAla Thr Ser Ala Phe Gln Thr Gln Lys 625 630 635 640 Pro Leu Lys Ser ThrSer Leu Ser Leu Phe Tyr Lys Lys Val Tyr Arg 645 650 655 Leu Ala Tyr LeuArg Leu Asn Thr Leu Cys Glu Arg Leu Leu Ser Glu 660 665 670 His Pro GluLeu Glu His Ile Ile Trp Thr Leu Phe Gln His Thr Leu 675 680 685 Gln AsnGlu Tyr Glu Leu Met Arg Asp Arg His Leu Asp Gln Ile Met 690 695 700 MetCys Ser Met Tyr Gly Ile Cys Lys Val Lys Asn Ile Asp Leu Lys 705 710 715720 Phe Lys Ile Ile Val Thr Ala Tyr Lys Asp Leu Pro His Ala Val Gln 725730 735 Glu Thr Phe Lys Arg Val Leu Ile Lys Glu Glu Glu Tyr Asp Ser Ile740 745 750 Ile Val Phe Tyr Asn Ser Val Phe Met Gln Arg Leu Lys Thr AsnIle 755 760 765 Leu Gln Tyr Ala Ser Thr Arg Pro Pro Thr Leu Ser Pro IlePro His 770 775 780 Ile Pro Arg Ser Pro Tyr Lys Phe Pro Ser Ser Pro LeuArg Ile Pro 785 790 795 800 Gly Gly Asn Ile Tyr Ile Ser Pro Leu Lys SerPro Tyr Lys Ile Ser 805 810 815 Glu Gly Leu Pro Thr Pro Thr Lys Met ThrPro Arg Ser Arg Ile Leu 820 825 830 Val Ser Ile Gly Glu Ser Phe Gly ThrSer Glu Lys Phe Gln Lys Ile 835 840 845 Asn Gln Met Val Cys Asn Ser AspArg Val Leu Lys Arg Ser Ala Glu 850 855 860 Gly Ser Asn Pro Pro Lys ProLeu Lys Lys Leu Arg Phe Asp Ile Glu 865 870 875 880 Gly Ser Asp Glu AlaAsp Gly Ser Lys His Leu Pro Gly Glu Ser Lys 885 890 895 Phe Gln Gln LysLeu Ala Glu Met Thr Ser Thr Arg Thr Arg Met Gln 900 905 910 Lys Gln LysMet Asn Asp Ser Met Asp Thr Ser Asn Lys Glu Glu Lys 915 920 925 393amino acids amino acid <Unknown> linear protein Protein 1..393 /note=“human p53” 9 Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro LeuSer Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu AsnAsn Val Leu 20 25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met LeuSer Pro Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro AspGlu Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala ProAla Ala Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp ProLeu Ser Ser Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr GlyPhe Arg Leu Gln 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val ThrCys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu AlaLys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr Pro Pro ProGly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys Gln Ser GlnHis Met Thr Glu Val Val Arg Arg Cys 165 170 175 Pro His His Glu Arg CysSer Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190 His Leu Ile Arg ValGlu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200 205 Arg Asn Thr PheArg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu 210 215 220 Val Gly SerAsp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser 225 230 235 240 SerCys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265270 Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro LysLys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile ArgGly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala LeuGlu Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser ArgAla His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr SerArg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp385 390

What is claimed is:
 1. A recombinant adenovirus expression vectorcomprising a partial or total deletion of a protein IX DNA and a geneencoding a foreign protein.
 2. The recombinant adenovirus expressionvector of claim 1, wherein the deletion of the protein IX gene sequenceextends from about 3500 bp from the 5′ viral termini to about 4000 bpfrom the 5′ viral termini.
 3. The recombinant adenovirus expressionvector of claim 2 further comprising deletion of a non-essential DNAsequence in adenovirus early region 3 and/or early region
 4. 4. Therecombinant adenovirus expression vector of claim 2 further comprisingdeletion of a DNA sequences designated adenovirus E1a and E1b.
 5. Therecombinant adenovirus expression vector of claim 2 further comprisingdeletion of early region 3 and/or 4 and DNA sequences designatedadenovirus E1a and E1b.
 6. The recombinant adenovirus expression vectorof claim 4 or 5 further comprising a deletion of up to forty nucleotidespositioned 3′ to the E1a and E1b and protein IX deletion and a foreignDNA molecule encoding a polyadenylation signal.
 7. The recombinantadenovirus expression vector of claims 1 to 6, wherein the adenovirus isa Group C adenovirus selected from a serotype 1, 2, 5 or
 6. 8. Therecombinant adenovirus expression vector of claim 1, wherein the gene isa DNA molecule up to 2.6 kilobases.
 9. The recombinant adenovirusexpression vector of claim 6, wherein the gene is a DNA molecule up to4.5 kilobases.
 10. The recombinant adenovirus expression vector of claim1, wherein the gene encodes a foreign functional protein or abiologically active fragment thereof.
 11. The recombinant adenovirusexpression vector of claim 10, wherein the gene encodes a foreignfunctional tumor suppressor protein or a biologically active fragmentthereof.
 12. The recombinant adenovirus expression vector of claim 1,wherein the gene encodes a suicide protein or functional equivalentthereof.
 13. A transformed host cell comprising the recombinantadenovirus expression vector of claim 1 or
 10. 14. The transformed hostcell of claim 13, wherein the host cell is a procaryotic or eucaryoticcell.
 15. A method for transforming a pathologic hyperproliferativemammalian cell comprising contacting the cell with the expression vectorof claim
 1. 16. A method of treating a pathology in an animal or mammalcaused by the absence of a tumor suppressor gene or the presence of apathologically mutated tumor suppressor gene comprising administering tothe animal or mammal an effective amount of the vector of claim 1containing a gene encoding a foreign functional protein having a tumorsuppressive function, under suitable conditions.
 17. The method of claim16, wherein the foreign protein is a functional tumor suppressorprotein.
 18. A method of gene therapy comprising administering to asubject an effective amount of the vector of claim
 1. 19. A method ofinhibiting the proliferation of a tumor in an animal comprisingadministering an effective amount of the adenoviral expression vector ofclaim 1 under suitable conditions to the animal.
 20. The method of claim19, wherein the gene encodes an anti-tumor agent.
 21. The method ofclaim 20, wherein the anti-tumor agent is a tumor suppressor gene. 22.The method of claim 20, wherein the anti-tumor agent is a suicide geneor functional equivalent thereof.
 23. The method of claim 21, whereinthe tumor is non-small cell lung cancer, small cell lung cancer,hepatocarcinoma, melanoma, retinoblastoma, breast tumor, colorectalcarcinoma, leukemia, lymphoma, brain tumor, cervical carcinoma, sarcoma,prostate tumor, bladder tumor, tumor of the reticuloendothelial tissues,Wilm's tumor, astrocytoma, glioblastoma, neuroblastoma, ovariancarcinoma, osteosarcoma, and renal cancer.
 24. The method of claim 19,wherein the vector is administered by intra-tumoral injection.
 25. Apharmaceutical composition comprising the recombinant adenoviralexpression vector of claim 1, 10 or
 12. 26. A method for reducing theproliferation of tumor cells in a subject comprising administering undersuitable conditions an effective amount of an adenoviral expressionvector of claim 12 and an effective amount of a thymidine kinasemetabolite or a functional equivalent thereof.
 27. The method of claim26, wherein the thymidine kinase metabolite is ganciclovir or6-methoxypurine arabinonucleoside or a functional equivalent thereof.28. The method of claim 26, wherein the adenoviral expression vector isadministered by injection into the tumor mass.
 29. The method of claim26, wherein the tumor cells are hepatocellular carcinoma.
 30. The methodof claim 29, wherein the adenoviral expression vector is administereddirectly into the hepatic artery of the subject.
 31. A kit for reducingthe proliferation of tumor cells comprising the components of theadenoviral expression vector of claim 12, a thymidine kinase metaboliteor functional equivalent thereof, pharmaceutical carriers andinstructions for the treatment of hepatocellular carcinoma using the kitcomponents.